Induced pluripotent cell-derived oligodendrocyte progenitor cells for the treatment of myelin disorders

ABSTRACT

The present invention relates to preparations of induced pluripotent cell-derived oligodendrocyte progenitor cells, and methods of making, isolating, and using these preparations.

This application is a divisional of U.S. patent application Ser. No.14/764,507, filed Jul. 29, 2015, which is a national stage applicationunder 35 U.S.C. §371 of PCT Application No. PCT/US2014/015019, filedFeb. 6, 2014, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 61/761,584, filed Feb. 6, 2013, and 61/780,265,filed Mar. 13, 2013, which are hereby incorporated by reference in theirentirety.

FIELD OF THE INVENTION

The present invention relates to preparations of induced pluripotentcell-derived oligodendrocyte progenitor cells, and methods of making,isolating, and using these cells.

BACKGROUND OF THE INVENTION

A broad range of diseases, from the inherited leukodystrophies tovascular leukoencephalopathies to multiple sclerosis, result from myelininjury or loss. In the pediatric leukodystrophies, in particular,compact myelin either fails to properly develop, or is injured in thesetting of toxic storage abnormalities. Recent studies have focused onthe use of transplanted oligodendrocytes or their progenitors for thetreatment of these congenital myelin diseases. Both rodent andhuman-derived cell implants have been assessed in a variety ofexperimental models of congenital dysmyelination. The myelinogenicpotential of implanted brain cells was first noted in the shiverer mouse(Lachapelle et al., “Transplantation of CNS Fragments Into the Brain ofShiverer Mutant Mice: Extensive Myelination by ImplantedOligodendrocytes,” Dev. Neurosci 6:325-334 (1983)). The shiverer is amutant deficient in myelin basic protein (MBP), by virtue of a prematurestop codon in the MBP gene that results in the omission of its last 5exons (Roach et al., “Chromosomal Mapping of Mouse Myelin Basic ProteinGene and Structure and Transcription of the Partially Deleted Gene inShiverer Mutant Mice,” Cell 42:149-155 (1985)). Shiverer is an autosomalrecessive mutation, and shi/shi homozygotes fail to develop centralcompact myelin. They die young, typically by 20-22 weeks of age, withataxia, dyscoordination, spasticity, and seizures. When fetal humanbrain tissue was implanted into shiverers, evidence of botholigodendrocytic differentiation and local myelination was noted(Lachapelle et al., “Transplantation of Fragments of CNS Into the Brainsof Shiverer Mutant Mice: Extensive Myelination by ImplantedOligodendrocytes,” Dev. Neurosci 6:326-334 (1983); Gumpel et al.,“Transplantation of Human Embryonic Oligodendrocytes Into ShivererBrain,” Ann NY Acad Sci 495:71-85 (1987); and Seilhean et al.,“Myelination by Transplanted Human and Mouse Central Nervous SystemTissue After Long-Term Cryopreservation,” Acta Neuropathol 91:82-88(1996)). However, these unfractionated implants yielded only patchyremyelination and would have permitted the co-generation of other,potentially undesired phenotypes. Enriched glial progenitor cells werethus assessed for their myelinogenic capacity, and were found able tomyelinate shiverer axons (Warrington et al., “Differential MyelinogenicCapacity of Specific Development Stages of the Oligodendrocyte LineageUpon Transplantation Into Hypomyelinating Hosts,” J. Neurosci Res34:1-13 (1993)), though with low efficiency, likely due to predominantlyastrocytic differentiation by the grafted cells. Yandava et al., “GlobalCell Replacement is Feasible via Neural Stem Cell Transplantation:Evidence from the Dysmyelinated Shiverer Mouse Brain,” Proc. Natl. Acad.Sci. 96:7029-7034 (1999), subsequently noted that immortalizedmultipotential progenitors could also contribute to myelination inshiverers. Duncan and colleagues similarly noted thatoligosphere-derived cells raised from the neonatal rodent subventricularzone could engraft another dysmyelinated mutant, the myelin-deficientrat, upon perinatal intraventricular administration (Learish et al.,“Intraventricular Transplantation of Oligodendrocyte Progenitors into aFetal Myelin Mutant Results in Widespread Formation of Myelin,” AnnNeurol 46:716-722 (1999)).

Human glial progenitor cells capable of oligodendrocytic maturation andmyelination have been derived from both fetal and adult human braintissue (Dietrich et al., “Characterization of A2B5+ Glial PrecursorCells From Cryopreserved Human Fetal Brain Progenitor Cells,” Glia40:65-77 (2002), Roy et al., “Identification, Isolation, andPromoter-Defined Separation of Mitotic Oligodendrocyte Progenitor CellsFrom the Adult Human Subcortical White Matter,” J. Neurosci.19:9986-9995 (1999), Windrem et al., “Fetal and Adult HumanOligodendrocyte Progenitor Cell Isolates Myelinate the CongenitallyDysmyelinated Brain,” Nat. Med. 10:93-97 (2004)), as well as from humanembryonic stem cells (Hu et al., “Differentiation of HumanOligodendrocytes From Pluripotent Stem Cells,” Nat. Protoc. 4:1614-1622(2009), Izrael et al., “Human Oligodendrocytes Derived From EmbryonicStem Cells: Effect of Noggin on Phenotypic Differentiation in Vitro andon Myelination in Vivo,” Mol. Cell. Neurosci. 34:310-323 (2007), andKeirstead et al., “Human Embryonic Stem Cell-Derived OligodendrocyteProgenitor Cell Transplants Remyelinate and Restore Locomotion AfterSpinal Cord Injury,” J. Neurosci. 25:4694-4705 (2005)) and have proveneffective in experimental models of both congenitally dysmyelinated (Simet al., “CD140a Identifies a Population of Highly Myelinogenic,Migration-Competent and Efficiently Engrafting Human OligodendrocyteProgenitor Cells,” Nat. Biotechnol. 29:934-941 (2011), Windrem et al.,“Fetal and Adult Human Oligodendrocyte Progenitor Cell IsolatesMyelinate the Congenitally Dysmyelinated Brain,” Nat. Med. 10:93-97(2004), Windrem et al., “Neonatal Chimerization With Human GlialProgenitor Cells Can Both Remyelinate and Rescue the Otherwise LethallyHypomyelinated Shiverer Mouse,” Cell Stem Cell 2:553-565 (2008)) andadult demyelinated (Windrem et al., “Progenitor Cells Derived From theAdult Human Subcortical White Matter Disperse and Differentiate asOligodendrocytes Within Demyelinated Lesions of the Rat Brain,” J.Neurosci. Res. 69:966-975 (2002)) brain and spinal cord. Yet thesesuccesses in immunodeficient mice notwithstanding, immune rejection hasthus far hindered the use of allogeneic human cells as transplantvectors. Concern for donor cell rejection has been especiallyproblematic in regards to the adult demyelinating diseases such asmultiple sclerosis, in which the inflammatory processes underlying thesedisorders can present an intrinsically hostile environment to anyallogeneic grafts (Keyoung and Goldman, “Glial Progenitor-Based Repairof Demyelinating Neurological Diseases,” Neurosurg. Clin. N. Am.18:93-104 (2007)).

The present invention is directed at overcoming this and otherdeficiencies in the art.

SUMMARY OF THE INVENTION

A first aspect of the present invention is directed to a preparation ofCD140a/PDGFRα positive cells where the preparation comprisesoligodendrocyte progenitor cells co-expressing OLIG2 and CD140a/PDGFRα,and where the preparation of cells is derived from pluripotential cellsderived from skin cells.

A second aspect of the present invention is directed to a preparation ofCD140a/PDGFRα positive cells where the preparation comprisesoligodendrocyte progenitor cells co-expressing OLIG2 and CD140a/PDGFRα,and where the preparation of cells is derived from pluripotential cellsderived from umbilical cord blood.

Another aspect of the present invention is directed to a preparation ofCD140a/PDGFRα positive cells where the preparation comprisesoligodendrocyte progenitor cells co-expressing OLIG2 and CD140a/PDGFRα,and where the preparation of cells is derived from pluripotential cellsderived from peripheral blood.

Another aspect of the present invention is directed to a preparation ofCD140a/PDGFRα positive cells where the preparation comprisesoligodendrocyte progenitor cells co-expressing OLIG2 and CD140a/PDGFRα,and where the preparation of cells is derived from pluripotential cellsderived from bone marrow.

Another aspect of the present invention is directed to a preparation ofcells at least ˜95% of which are CD140a/PDGFRα positive cells, where thepreparation comprises oligodendrocyte progenitor cells co-expressingOLIG2 and CD140a/PDGFRα, and where the oligodendrocyte progenitor cellsretain one or more epigenetic markers of a differentiated somatic cellother than an oligodendrocyte.

Another aspect of the present invention is directed to a method ofproducing an enriched preparation of oligodendrocyte progenitor cells.This method involves culturing a population of induced pluripotent stemcells under conditions effective for the cells to form embryoid bodies,and inducing cells of the embryoid bodies to differentiate intoneuroepithelial cells and form neuroepithelial cell colonies. The methodfurther involves exposing the neuroepithelial cell colonies toconditions effective to induce differentiation to oligodendrocyteprogenitor cells, thereby forming an enriched preparation ofoligodendrocyte progenitor cells co-expressing OLIG2 and CD140a/PDGFRα.

Another aspect of the present invention is directed to a method oftreating a subject having a condition mediated by a loss of myelin or aloss of oligodendrocytes that involves administering to the subject anyone of the cell preparations of the present invention under conditionseffective to treat the condition.

To date a robust protocol for the scalable production of enriched and/orpurified preparation of myelinogenic oligodendrocytes from inducedpluripotential cells (iPSCs) has not been reported. Pouya et al., “HumanInduced Pluripotent Stem Cells Differentiation into OligodendrocyteProgenitors and Transplantation in a Rat Model of Optic ChiasmDemyelination,” PLoS ONE 6(11):e27925 (2011) (“Pouya”), which is herebyincorporated by reference in its entirety, reports the production ofhuman iPSc-derived oligodendrocyte progenitor cells; however theembryonic stem cell based differentiation protocol utilized by Pouya wasnot tailored to specifically and controllably direct neural progenitorcell differentiation followed by oligodendrocyte progenitor cellproduction. The lack of controlled differentiation in Pouya's protocolresults in random differentiation and the generation of a heterogenouscell preparation contaminated with pluripotent cell types (e.g., Oct4,SOX2 expressing cells), not fully differentiated cell types (e.g., Pax6and Tuj1 expressing cells), and differentiated non-oligodendrocyteprogenitor cell types (e.g., neurons, astrocytes, as well as non-neuralcell types). Additionally, because of random differentiation, cellsorting or selection techniques based on a single marker such asCD140a/PDGFRα, which is only specific for oligodendrocyte progenitorcells within a mixed population of brain cells but not a mixedpopulation of non-brain cells, cannot be used to reliably enrich orpurify the desired oligodendrocyte progenitor cells from Pouya'spreparation. Accordingly, the preparation of Pouya is not clinically ortherapeutically useful for treating conditions arising from the loss ofoligodendrocytes or loss of myelin.

As described herein, applicants have developed a robust and reliableprotocol for the scalable production of highly enriched preparations ofmyelinogenic oligodendrocytes from skin-derived human iPSCs. Asdemonstrated herein, the iPSCs reliably progress through serial stagesof neural progenitor, glial progenitor cell, oligodendrocyte, andastrocyte differentiation in vitro. Since random differentiation isavoided, the CD140a/PDGFRα⁺ cells of the population are oligodendrocyteprogenitor cells, which can be further purified and enriched usingCD140a/PDGFRα based sorting prior to clinical use. The myelinationcompetence of the hiPSC-derived oligodendrocyte progenitor cells of thepresent invention was assessed in the shiverer mouse model, a geneticmodel of congenital hypomyelination. These cells efficiently androbustly myelinate the hypomyelinated shiverer brain, with no evidenceof either tumorigenesis or heterotopic non-glial differentiation. Thetransplanted animals survived significantly longer than their untreatedcounterparts, and the majority were frankly spared otherwise earlylethality, a striking clinical rescue of a fatal hereditary disorder viaan iPSC-based strategy. Accordingly, under the proper conditions, humaniPSCs are a feasible and effective source of oligodendrocyte progenitorcells and their derived myelinogenic central oligodendrocytes aresuitable for use as patient-specific therapeutic vectors for treatingdisorders of central myelin.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1P show that human iPSCs (hiPSCs) can be directed intooligodendrocyte progenitor cell (OPC) fate. FIG. 1A is schematicprotocol for directed differentiation of hiPSCs into OPCS. Embryoidbodies (EBs) were differentiated from undifferentiated hiPSCs (stage 1)from DIV 0-5. EBs were then differentiated as neuroepithelial (NE) cellsin neural induction media (NIM; see Materials and Methods for Examples1-9) with bFGF. FIGS. 1B-1D show that the undifferentiated hiPSCs(stage 1) and hiPSC colonies expressed the pluripotency markers SSEA4and OCT4. Phase contrast (FIG. 1B); SSEA4 (red) (FIG. 1C); DAPI (blue).OCT4 (green) (FIG. 1D); DAPI (blue). Erythroid bodies (EBs) (FIG. 1E)and neuroepithelial cells (FIGS. 1F and 1G) could be generated fromhiPSCs (stages 2 and 3). hiPSC-derived neuroepithelial cells at thisstage expressed the neuroepithelial markers PAX6 and SOX1. Phasecontrast (FIG. 1E); PAX6 (green) (FIG. 1F); SOX1 (red) (FIG. 1G). FIGS.1H and 1I demonstrate that OLIG2⁺ and NKX2.2⁻ early glial progenitorcells appeared under the influence of retinoic acid (RA) andpurmorphamine, a small-molecule agonist of sonic hedgehog signaling. Bystage 4, OLIG2 was expressed in early pre-OPCS, which then seriallydeveloped NKX2.2 expression. OLIG2 (red) (FIG. 1H); NKX2.2 (green) (FIG.1I). FIG. 1J show OLIO2⁺/NKX2.2 early pre-OPCS were differentiated intolater-stage OLIG2⁺/NKX2.2⁺ pre-OPCS when RA was replaced by bFGF atstage 5. OLIG2 (red); NKX2.2 (green). FIG. 1K-1M show that pre-OPCS werefurther differentiated into bipotential OPCS in glial induction media(GIM; see Materials and Methods for Examples 1-9) supplemented withPDGF-AA, T3, NT3, and IGF. Stage 6 was extended as long as 3-4 monthsfor maximization of the production of myelinogenic OPCS. By the time oftransplant, these cells expressed not only OLIG2 and NKX2.2 (FIG. 1K),but also SOX10 (FIG. 1L) and PDGFRα(FIG. 1M). By the end of stage 6,hiPSC OPCS could be identified as OLIG2⁺/NKX2.2⁺/SOX10⁺/PDGFRα⁺. OLIG2(red), NKX2.2 (green) (FIG. 1K); SOX10 (red), NKX2.2 (green) (FIG. 1L);PDGFRα (red), OLIG2 (green) (FIG. 1M). FIG. 1N-1P shows in vitroterminal differentiation of hiPSC OPCS into hiPSC-derivedoligodendrocytes (hiOLs), identified by O4⁺ (FIG. 1N) and MBP⁺ (FIG.1O). OLs and GFAP⁺ astrocytes (FIG. 1P) arose with reduction in glialmitogens. 04 (green). (FIG. 1N); MBP (red) (FIG. 1O); GFAP (green) (FIG.1P); DAPI (blue). Scale: 100 μm (FIGS. 1B-1N, 1P) and 25 μm (FIG. 1O).

FIG. 2 shows the characterization of hiPSC lines. All three hiPSC linesin this study show hESC-like morphology (phase images), when compared tothe hESC line WA09/H9 (top row). Immunolabeling confirmed that thehiPSCs expressed NANOG, OCT4, SOX2, SSEA4 and TRA-1-60immunoreactivities. Scale: 200 μm.

FIGS. 3A-3J demonstrate that both astrocytes and oligodendroctyes areefficiently generated from hiPSC OPCs. FIG. 3A shows the expression ofneural markers during induction of oligodendroglial-lineagehiPSC-derived neuroepithelial cells in stage 3, pre-OPCs in stages 4 and5, and OPCs in stage 6. Cultures were immunostained for PAX6 and SOX1,or OLIG2 and NKX2.2, respectively. The proportion of immunopositiveclusters for each marker set were scored for each hiPSC line. At leastthree repeats in each group were performed; data are provided asmeans±SEM. In stage 6, gliogenic clusters were dissociated tosingle-cell suspensions and plated in GIM, resulting in the terminaldifferentiation of both astrocytes and myelinogenic OLs. FIGS. 3B and 3Cdemonstrate that GFAP⁺ astrocytes were evident in cultures of hiPSC OPCsby 95 DIV; C27-derived (FIG. 3B) and K04-derived (FIG. 3C) astrocytesare shown here. FIG. 3D is a graph showing the proportion of GFAP⁺astrocytes among all cultured cells at 95 DIV; the remainder expressedoligodendroglial lineage markers (means±SEM; see FIG. 4I). ICC,immunocytochemistry. FIGS. 3E-3J are images taken later in stage 6 (160DIV), hiPSC-derived OPCs differentiated as both O4⁺ (FIGS. 3E and 3F)and MBP⁺ (FIG. 3G) OLs. FIGS. 3H-3J show that when cocultured with humanfetal cortical neurons, hiPSC OPCs derived from C27 (FIG. 3H), C14 (FIG.3I), and K04 (FIG. 3J) hiPSCs all generated MBP⁺ myelinogenic OLs thatengaged NF⁺ axons (MBP, red; NF, green). Scale: 50 μm.

FIGS. 4A-4M show serial neuroepithelial and glial differentiation fromhiPSCs. FIG. 4A shows that neuroepithelial cells could be efficientlyinduced from both the keratinocyte-derived K04 hiPSC line and thefibroblast-derived C14 and C27 hiPSC lines. By stage 3, neural stemcells were evident and organized in rosette-like structures, andco-expressed the proneural markers, PAX6 and SOX1 (panel of cell imagelabeled FIG. 4A). FIGS. 4B-4C show early (Stage 4; FIG. 4B) pre-OPCs andlater (Stage 5: FIG. 4C) OPCs could be induced from all 3 hiPSC (C14,C27 and K04) lines tested, as well as from H9 hESC (WA09) cells. ByStage 4, most hiPSC- or hESC-derived pre-OPCs expressed OLIG2; fewerexpressed NKX2.2. By Stage 5, more NKX2.2⁺ pre-OPCs appeared, such thatdouble-labeled OLIG2⁺/NKX2.2⁺ cells typically comprised 70-90% of allDAPI⁺ cells in each line assessed. FIGS. 4D-4H show that neuroepithelialcells from each hiPSC and hESC line could be reliably differentiatedinto OLIG2⁺/NKX2.2⁺/SOX10⁺ OPCs. FIGS. 4E-4F are single color splits ofFIG. 4D. FIG. 4I is a bar graph showing the proportions of OLIG2, NKX2.2or SOX10 expressing OPCs quantified in K04 iPSC OPCs at stage 6. Scale:FIGS. 4A-4H, 50 μm. FIGS. 4J-4M are bar graphs showing thatoligodendrocyte progenitor differentiation occurred concurrently withdepletion of transcripts associated with pluripotentiality includingOCT4 (FIG. 4J), hTERT (FIG. 4K), OLIG2 (FIG. 4L) or NKX2.2 (FIG. 4M).mRNAs from undifferentiated (stage 1) hiPSCs were compared to thoseextracted from differentiated (stage 6) iPSC hOPCs, derived from C27,K04 and WA9/H9 cells. Whereas by stage 6 both OCT4 and hTERT transcriptswere significantly down-regulated in hiPSC OPCS, thepro-oligodendroglial transcripts OLIG2 and NKX2.2 were significantlyup-regulated. Data are represented as means±SEM.

FIGS. 5A-5C show that OPCS can be isolated from mixed hiPSC culture byCD140a- and CD9-Directed FACS. As shown in FIG. 5A, hiPSC OPC-derivedOLs were recognized and isolated by FACS using monoclonal antibody O4,which recognizes oligodendrocytic sulfatide. The incidence of O4⁺oligodendroglia varied across different hiPSC lines, from 4% to 12% (seeTable 3; n=4-7 experiments). FIG. 5B shows that OPCS derived from hiPSCs(C27 and K04) were readily recognized with the cell-surface marker A2B5.FIG. 5C shows that OPCS derived from either hiPSCs (C27 and K04) orhESCs (WA09/H9) were readily recognized with cell-surface markers,PDGFRα (CD140a), and CD9 by FACS analysis. The relative proportions ofCD140a, CD9, and CD140a/CD9 double-labeled cells varied across thedifferent cell-line-derived OPCS (n=4-7 experiments).

FIGS. 6A-6G demonstrate that hiPSCs migrate widely and differentiate asastroglia and myelinogenic OLs. hiPSC OPCS generated from all threehiPSC lines migrated throughout the shiverer brain, engrafting mostdensely in white matter. Distributions of C27 (FIG. 6A) and K04 (FIG.6B) hiPSC-derived OPCS are shown (hNA⁺, red, mapped in StereoInvestigator). By 13 weeks of age, C27 hiPSC OPCS (FIG. 6C), K04 hiPSCOPCS (FIGS. 6E and 6G), and C14 hiPSC OPCS (FIG. 6H) matured intoMBP-expressing oligodendroglia (green) throughout the subcortical whitematter, including callosal and capsular (FIGS. 6C, 6E, and 6H) as wellas striatal (FIG. 6G) tracts. In these 13-week-old shiverer mouserecipients, C27 (FIG. 6D), K04 (FIG. 6F), and C14 (FIG. 6I)hiPSC-derived OPCS also differentiated as astroglia (human-specificGFAP, green), especially as fibrous astrocytes in the central whitematter. Scale: 100 μm (FIG. 6C-6I).

FIGS. 7A-7K show that hiPSC OPCS robustly myelinate in vivo. Confocalimages of the callosal and capsular white matter of mice engrafted withhiPSC OPCS derived from all three tested hiPSC lines demonstrate densedonor-derived myelination: C27-derived (FIGS. 7A and 7B), K04-derived(FIGS. 7E-7G), and C14-derived (FIGS. 71 and 7J). FIGS. 7A, 7G, and 7Jshow abundant, donor-derived MBP expression (green) by C27, K04, and C14hiPSC OPCs (hNA, red), respectively. Representative z stacks ofindividual hNA⁺ OLs are shown as asterisks in (FIGS. 7A and 7E). By the19 week time point assessed here, C27 (FIG. 7B), K04 (FIGS. 7F and 7G),and C14 (FIG. 7J) hiPSC oligodendroglia robustly myelinated axons (NF,red). hiPSC-derived oligodendroglial morphologies are exemplified inFIG. 7F (K04) and FIG. 7I (C14); FIG. 7F shows multiaxon myelination bysingle OLs in the striatum. hiPSC OPCs also generated astroglia (FIG.7C, C27; FIG. 7H, K04), which exhibited the complex fibrous morphologiestypical of human astrocytes (human-specific GFAP, green). Many cellsalso remained as progenitors, immunostaining for NG2 (FIG. 7D, C27) andhuman-specific PDGFRα (FIG. 7K, C14).Scale: 50 μm (FIG. 7A-7C, 7G, 7J);20 μm (FIGS. 7C-7F, 7H, 7K); and 10 μm (FIG. 7I, insets to 7A and 7E).

FIGS. 8A-8E show that hiPSC OPCs myelinate widely to greatly extend thesurvival of hypomyelinated mice. FIG. 8A is a dot map indicating thedistribution of hiPSC-derived donor cells (C27) at 7 months of age,following neonatal engraftment in a shiverer mouse brain. Widespreaddispersal and chimerization by hiPSC OPCs is evident (hNA, red). FIG. 8Bshows hiPSC-OPC-derived myelination in a shiverer forebrain at 7 months;section 1 mm lateral to section of FIG. 8A. MBP immunoreactivity (green)is all donor derived. FIGS. 8C and 8D show myelination in sagittalsections taken at different mediolateral levels from two additional7-month-old mice, each engrafted with C27 hiPSC OPCs at birth. FIG. 8Eis a Kaplan-Meier plot showing the survival of C27 iPSC-OPC-implanted(n=22) versus saline-injected (n=19) control mice. Remaining engraftedmice sacrificed for electron microscopy at 9-10 months (>270 days).Scale: 2 mm (FIGS. 8A and 8B).

FIG. 9 shows widespread myelination by K04-Derived hiPSC OPCsMyelination by K04-derived hiPSC OPCs in coronal sections ofneonatally-engrafted shiverer brain, at 4.5 months of age. MBP, green.Scale: 2 mm.

FIGS. 10A-10I show hiPSC-derived oligodendrocytes produce compact myelinand induce Nodes of Ranvier. Representative electron microscopic imagesof sections through the corpus callosum (FIGS. 10A and 10B) and ventralpons (FIG. 10C) of a 40-week-old shiverer mouse neonatally engraftedwith C27 hiPSC OPCs, showing donor-derived compact myelin with evidentmajor dense lines, ensheathing mouse axons. FIGS. 10D-10G showhigher-power images of donor-derived myelin in the corpus callosum, alsoat 40 weeks. FIGS. 10D and 10E show that the alternating major dense(arrowheads) and intraperiod lines, characteristic of mature myelin, areevident. In FIGS. 10F and 10G, myelin sheaths in the corpus callosumensheathing central axons are shown, their maturation manifested byparallel arrays of tight junctions (FIG. 10F, arrowhead) and major denselines (FIG. 10G, arrowhead). This mature myelination permitted theorganization of architecturally appropriate nodes of Ranvier by hiPSColigodendroglia. In FIG. 10H and FIG. 10I, nodal reconstitution intransplanted shiverers is demonstrated by immunostaining ofoligodendrocytic paranodal Caspr protein (red), seen flanking nodes ofRanvier identified here by βIV spectrin (green). An isolated node isshown in confocal cross-section in FIG. 10I. Scale: 200 nm (FIG.10A-10E); 100 nm (FIGS. 10F and 10G); and 5 μm (FIGS. 10H and 10I).

FIGS. 11A-11E show myelination in hiPSC-OPC-transplanted but not inuntreated Shiverer mice. Toluidine blue stained semi-thin sectionsthrough the corpus callosum and cortical layer VI in C27 hiPSC-derivedOPC-transplanted (FIG. 11A) or untreated (FIG. 11B) shiverer brain.FIGS. 11C and 11D are electron micrographs of the callosum oftransplanted (FIG. 11C) and untreated (FIG. 11D) shiverer brain. FIG.11E is a lower magnification view of untransplanted shiverer whitematter; same animal as shown in FIG. 11D. Scale: FIGS. 11A-11B, 10 μm;FIGS. 11C-11D, 500 nm; FIG. 11E, 1 μm.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to preparations of CD140a/PDGFRαpositive cells where the preparation comprises oligodendrocyteprogenitor cells co-expressing OLIG2 and CD140a/PDGFRα. In one aspect ofthe present invention, the preparation of cells is derived frompluripotential cells that are derived from skin cells. In another aspectof the present invention, the preparation of cells is derived frompluripotential cells that are derived from umbilical cord blood. Inanother aspect of the present invention, the preparation of cells isderived from pluripotential cells that are derived from peripheralblood. In another aspect of the present invention, the preparation ofcells is derived from pluripotential cells that are derived from bonemarrow.

Oligodendrocyte progenitor cells, as referred to herein, comprise apopulation of bipotential progenitor cells that can give rise to botholigodendrocytes and astrocytes.

As described herein, the preparation of CD140a/PDGFRα positive cells arederived from pluripotential cells using a robust and scalable protocolthat directs a controlled differentiation process. In one embodiment ofthe present invention, the pluripotential cells are induced pluripotentstem cells (iPSC). “Induced pluripotent stem cells” as used hereinrefers to pluripotent cells that are derived from non-pluripotent cells,such as somatic cells or tissue stem cells. For example, and withoutlimitation, iPSCs can be derived from adult fibroblasts (see e.g.,Streckfuss-Bomeke et al., “Comparative Study of Human-InducedPluripotent Stem Cells Derived from Bone Marrow Cells, HairKeratinocytes, and Skin Fibroblasts,” Eur. Heart J. doi:10.1093/eurheartj/ehs203 (2012), which is hereby incorporated byreference in its entirety), umbilical cord blood (see e.g., Cai et al.,“Generation of Human Induced Pluripotent Stem Cells from Umbilical CordMatrix and Amniotic Membrane Mesenchymal Cells,” J. Biol. Chem. 285(15):112227-11234 (2110) and Giorgetti et al., “Generation of InducedPluripotent Stem Cells from Human Cord Blood Cells with only TwoFactors: Oct4 and Sox2,” Nature Protocols, 5(4):811-820 (2010), whichare hereby incorporated by reference in their entirety), bone marrow(see e.g., Streckfuss-Bomeke et al., “Comparative Study of Human-InducedPluripotent Stem Cells Derived from Bone Marrow Cells, HairKeratinocytes, and Skin Fibroblasts,” Eur. Heart J. doi:10.1093/eurheartj/ehs203 (Jul. 12, 2012), and Hu et al., “EfficientGeneration of Transgene-Free Induced Pluripotent Stem Cells from Normaland Neoplastic Bone Marrow and Cord Blood Mononuclear Cells,” Blood doi:10.1182/blood-2010-07-298331 (Feb. 4, 2011) which are herebyincorporated by reference in their entirety), and peripheral blood (seee.g., Sommer et al., “Generation of Human Induced Pluripotent Stem Cellsfrom Peripheral Blood using the STEMCCA Lentiviral Vector,” J. Vis. Exp.68: e4327 doi:10.3791/4327 (2012), which is hereby incorporated byreference in its entirety). iPSCs can also be derived fromkeratinocytes, mature B cells, mature T cells, pancreatic β cells,melanocytes, hepatocytes, foreskin cells, cheek cells, lung fibroblasts,myeloid progenitors, hematopoietic stem cells, adipose-derived stemcells, neural stem cells, and liver progenitor cells. Methods ofgenerating iPSCs from non-pluripotent cells are described in more detailherein.

Within the brain, PDGFRα is highly and specifically expressed byoligodendrocyte progenitor cells (Sim et al., “CD140a Identifies aPopulation of Highly Myelinogenic Migration-Competent and EfficientlyEngrafting Human Oligodendrocyte Progenitor Cells,” Nat. Biotech.29(10): 934-941 (2011), which is hereby incorporated by reference in itsentirety). CD140a is an ectodomain of the PDGFRα that can be readilydetected and, therefore, serves as a reliable indicator or marker ofPDGFRα expression. Thus, PDGFRα positive cells can be identified byCD140a and a population of PDGFRα cells can be enriched using CD140a.Such cells are referred to as PDGFα⁺/CD140a⁺ cells.

While PDGFRα is a highly specific marker of oligodendrocyte progenitorcells within a population of brain-derived cells, PDGFRα is expressed bya number of cells outside of the brain, and, therefore, does notconstitute an oligodendrocyte specific marker in the context of a mixedpopulation of cells containing both brain and non-brain cell types. Dueto the pluripotent nature of iPSCs and possible random differentiationof these cells, the use of more than one oligodendrocyte marker isdesirable for accurate identification of oligodendrocyte progenitorcells within a preparation of cells derived from iPSCs. Accordingly, theoligodendrocyte progenitor cells of the various cell preparations of thepresent invention are identified by their co-expression of CD140a/PDGFRαand oligodendrocyte transcription factor 2 (OLIG2). In some embodimentsof the present invention, the oligodendrocyte progenitor cells of thepreparation are further or alternatively identified by co-expression ofone or more other oligodendrocyte progenitor cell markers such as SOX10,CD9, NKX2.2, or any combination thereof (see e.g., U.S. PatentPublication No. 2011/0059055 to Goldman et al., which is herebyincorporated by reference in its entirety).

The CD140a/PDGFRα/OLIG2 oligodendrocyte progenitor cell fraction of apreparation of the present invention may constitute greater than 30% ofthe preparation. In another embodiment of the present invention, theoligodendrocyte progenitor cell fraction of the preparation constitutesgreater than 40% of the preparation. In other embodiments of the presentinvention, the oligodendrocyte progenitor cell fraction of thepreparation constitutes >45% of the preparation, >50% of thepreparation, >55% of the preparation, >60% of the preparation, >65% ofthe preparation, >70% of the preparation, >75% of the preparation, >80%of the preparation, or >90% of the preparation.

Another aspect of the present invention relates to a preparation ofcells that has been enriched for oligodendrocyte progenitor cells. Thisaspect of the present invention is directed to a preparation of cells,at least 95% of which are CD140a/PDGFRα positive cells, where thepreparation comprises oligodendrocyte progenitor cells co-expressingOLIG2 and CD140a/PDGFRα, and where the oligodendrocyte progenitor cellsretain one or more epigenetic markers of a differentiated somatic cellother than an oligodendrocyte. In one embodiment of the presentinvention, greater than 95% of the cells in the preparation areCD140a/PDGFRα positive cells. In another embodiment of the presentinvention, greater than 98% of the cells in the preparation areCD140a/PDGFRα positive cells. In one embodiment of this aspect of thepresent invention, at least 90% of the cell preparation comprisesoligodendrocyte progenitor cells. In another embodiment of this aspectof the present invention, at least 95% of the cell preparation comprisesoligodendrocyte progenitor cells.

The cell preparations of the present invention are preferablysubstantially free of non-oligodendrocyte progenitor cell contaminants.In particular, preparations of CD140a/PDGFRα positive cells aresubstantially free (e.g., containing less than 20%, 15%, 10%, 9%, 8%,7%, 6%, 5%, 4%, 3%, 2%, or 1%) of other neural cell types such asastrocytes (e.g., GFAP antibody defined cells), neurons (e.g., MAP2 andPSA-NCAM antibody defined cells), microglia (e.g., CD11, CD32, and CD36antibody defined cells), or non-brain cell types. The cell preparationsof the present invention containing oligodendrocyte progenitor cells arealso substantially free (e.g., containing less than 20%, 15%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%) of non-differentiated, residualpluripotent cell types, e.g., the preparation is substantially free ofcells expressing either OCT4, NANOG, or SSEA4, and is substantially freeof less differentiated cell lineages, e.g., neural progenitor cellsidentified by PAX6 and/or TUJ1 expression.

In one embodiment of the present invention, the cells of the preparationof the present invention are mammalian cells, including, for example,but without limitation, human, monkey, rat, or mouse cells.

The cell preparations of the present invention, and in particular, theoligodendrocyte progenitor cell populations of the present invention canbe structurally distinguished from tissue derived or embryonic stemcell-derived oligodendrocyte progenitor cell counterparts based on themaintenance of one or more epigenetic markers of its somatic cellorigin. For example, when the iPSCs are derived from skin cells such asdermal fibroblasts, the iPSC-derived oligodendrocyte progenitor cellsmaintain one or more epigenetic markers of a somatic skin cell. The oneor more epigenetic markers may include, without limitation, methylationmarks (e.g., DNA and/or RNA methylation markers), or a histonemodification (e.g., acetylation, methylation, ubiquitylation,phosphorylation, or sumoylation).

The oligodendrocyte progenitor cell preparations of the presentinvention are functionally distinguishable from their tissue derived orembryonic stem cell-derived oligodendrocyte progenitor cellcounterparts. As demonstrated herein the cell preparations of thepresent invention have an in vivo myelination efficiency that is greaterthan the in vivo myelination efficiency of a preparation ofA2B5⁺/PSA-NCAM⁻ or CD140a⁺ sorted fetal human tissue derivedoligodendrocyte progenitor cells. Myelination efficiency is measured bythe proportion of central axons myelinated as a function of time afterengraftment. As demonstrated herein, the cell preparations of thepresent invention are also capable of achieving an in vivo myelinationdensity upon engraftment which is greater than that achieveable with apreparation of A2B5⁺/PSA-NCAM⁻ or CD140a⁺ sorted fetal human tissuederived oligodendrocyte progenitor cells. Additionally, the cellpreparations of the present invention are capable, upon engraftment, ofachieving improved survival in a myelination deficient mammal comparedto that achieveable with a preparation of A2B5⁺/PSA-NCAM⁻ or CD140a⁺sorted fetal human tissue derived oligodendrocyte progenitor cells. Inother words, the proportion of myelination deficient mammals survivingat any given timepoint is greater in iPSC oligodendrocyte progenitorengrafted mammals than fetal human tissue derived A2B5⁺/PS-NCAM⁻ cellengrafted mammals that had otherwise been treated identically. Forexample, only ˜25% of animals engrafted with fetal tissue derivedoligodendrocyte progenitor cells survive beyond 6 months of age, whereas50% of animals engrafted with iPSC-derived oligodendrocyte progenitorcells survive beyond 6 months. Accordingly, the oligodendrocyteprogenitor cell preparations of the present invention are functionallydistinguishable from non-iPSC derived oligodendrocyte progenitor cellpreparations.

The cell preparations of the present invention, including theCD140a/PDGFRα enriched preparations can be optionally expanded inculture to increase the total number of cells. The cells can be expandedby either continuous or pulsatile exposure to PDGF-AA or AB as mitogensthat support the expansion of oligodendrocyte progenitor cells; they canbe exposed to fibroblast growth factors, including FGF2, FGF4, FGF8 andFGF9, which can support the mitotic expansion of the glial progenitorcells, but which can bias their differentiation to a mixed population ofastrocytes as well as oligodendrocytes. The cells can also be expandedin media supplemented with combinations of FGF2, PDGF, and NT3, whichcan optionally be supplemented with either platelet-depleted or wholeserum (see Nunes et al. “Identification and Isolation of MultipotentNeural Progenitor Cells from the Subcortical White Matter of the AdultHuman Brain,” Nature Medicine 9:239-247; Windrem et al., “Fetal andAdult Human Oligodendrocyte Progenitor Cell Isolates Myelinate theCongenitally Dysmyelinated Brain,” Nature Medicine 10:93-97 (2004),which are incorporated by reference for the methods and compositionsdescribed therein).

The populations of oligodendrocyte progenitor cells are optionallygenetically modified to express one or more proteins of interest. Forexample, the cells can be modified to express an exogenous targetingmoiety, an exogenous marker (for example, for imaging purposes), or thelike. The oligodendrocyte progenitor cells of the cell preparations ofthe present invention can be optionally modified to overexpress anendogenous targeting moiety, marker, or a myelin basic protein, or thelike.

Another aspect of the present invention is directed to a method ofproducing an enriched preparation of oligodendrocyte progenitor cells.This method involves culturing a population of induced pluripotent stemcells under conditions effective for the cells to form embryoid bodies,and inducing cells of the embryoid bodies to differentiate intoneuroepithelial cells and form neuroepithelial cell colonies. The methodfurther involves exposing the neuroepithelial cell colonies toconditions effective to induce differentiation to oligodendrocyteprogenitor cells, thereby forming an enriched preparation ofoligodendrocyte progenitor cells co-expressing OLIG2 and CD140a/PDGFRα.The enriched preparation of oligodendrocyte progenitor cells may furtherexpress SOX10, CD9 or a combination thereof.

iPSCs can be derived from any species, including but not limited to,human, non-human primates, rodents (mice, rats), ungulates (cows, sheep,etc), dogs, cats, rabbits, hamsters, goats, and the like. The iPSCs canbe obtained from embryonic, fetal, newborn, and adult tissue, fromperipheral blood, umbilical cord blood, and bone marrow. Exemplarysomatic cells that can be used include fibroblasts, such as dermalfibroblasts obtained by a skin sample or biopsy, synoviocytes fromsynovial tissue, keratinocytes, mature B cells, mature T cells,pancreatic β cells, melanocytes, hepatocytes, foreskin cells, cheekcells, or lung fibroblasts. Exemplary stem or progenitor cells that aresuitable for iPSC production include, without limitation, myeloidprogenitors, hematopoietic stem cells, adipose-derived stem cells,neural stem cells, and liver progenitor cells. Although skin and cheekprovide a readily available and easily attainable source of appropriatecells, virtually any cell can be used.

Induced pluripotent stem cells can be produced by expressing acombination of reprogramming factors in a somatic cell. Suitablereprogramming factors that promote and induce iPSC generation includeone or more of Oct4, Klf4, Sox2, c-Myc, Nanog, C/EBPα, Esrrb, Lin28, andNr5a2. In certain embodiments, at least two reprogramming factors areexpressed in a somatic cell to successfully reprogram the somatic cell.In other embodiments, at least three reprogramming factors are expressedin a somatic cell to successfully reprogram the somatic cell. In otherembodiments, at least four reprogramming factors are expressed in asomatic cell to successfully reprogram the somatic cell.

iPSCs may be derived by methods known in the art including the useintegrating viral vectors (e.g., lentiviral vectors, induciblelentiviral vectors, and retroviral vectors), excisable vectors (e.g.,transposon and floxed lentiviral vectors), and non-integrating vectors(e.g., adenoviral and plasmid vectors) to deliver the genes that promotecell reprogramming (see e.g., Takahashi and Yamanaka, Cell 126:663-676(2006); Okita. et al., Nature 448:313-317 (2007); Nakagawa et al., Nat.Biotechnol. 26:101-106 (2007); Takahashi et al., Cell 131:1-12 (2007);Meissner et al. Nat. Biotech. 25:1177-1181 (2007); Yu et al. Science318:1917-1920 (2007); Park et al. Nature 451:141-146 (2008); and U.S.Patent Application Publication No. 2008/0233610, which are herebyincorporated by reference in their entirety). Other methods forgenerating IPS cells include those disclosed in WO2007/069666,WO2009/006930, WO2009/006997, WO2009/007852, WO2008/118820, U.S. PatentApplication Publication Nos. 2011/0200568 to Ikeda et al., 2010/0156778to Egusa et al., 2012/0276070 to Musick, and 2012/0276636 to Nakagawa,Shi et al., Cell Stem Cell 3(5): 568-574 (2008), Kim et al., Nature 454:646-650 (2008), Kim et al., Cell 136(3):411-419 (2009), Huangfu et al.,Nature Biotechnology 26: 1269-1275 (2008), Zhao et al., Cell Stem Cell3: 475-479 (2008), Feng et al., Nature Cell Biology 11: 197-203 (2009),and Hanna et al., Cell 133(2): 250-264 (2008) which are herebyincorporated by reference in their entirety.

Integration free approaches, i.e., those using non-integrating andexcisable vectors, for deriving iPSCs free of transgenic sequences areparticularly suitable in the therapeutic context. Suitable methods ofiPSC production that utilize non-integrating vectors include methodsthat use adenoviral vectors (Stadtfeld et al., “Induced Pluripotent StemCells Generated without Viral Integration,” Science 322: 945-949 (2008),and Okita et al., “Generation of Mouse Induced Pluripotent Stem Cellswithout Viral Vectors,” Science 322: 949-953 (2008), which are herebyincorporated by reference in their entirety), Sendi virus vectors(Fusaki et al., “Efficient Induction of Transgene-Free Human PluripotentStem Cells Using a Vector Based on Sendi Virus, an RNA Virus That DoesNot Integrate into the Host Genome,” Proc Jpn Acad. 85: 348-362 (2009),which is hereby incorporated by reference in its entirety),polycistronic minicircle vectors (Jia et al., “A Nonviral MinicircleVector for Deriving Human iPS Cells,” Nat. Methods 7: 197-199 (2010),which is hereby incorporated by reference in its entirety), andself-replicating selectable episomes (Yu et al., “Human InducedPluripotent Stem Cells Free of Vector and Transgene Sequences,” Science324: 797-801 (2009), which is hereby incorporated by reference in itsentirety). Suitable methods for iPSC generation using excisable vectorsare described by Kaji et al., “Virus-Free Induction of Pluripotency andSubsequent Excision of Reprogramming Factors,” Nature 458: 771-775(2009), Soldner et al., “Parkinson's Disease Patient-Derived InducedPluripotent Stem Cells Free of Viral Reprogramming Factors,” Cell136:964-977 (2009), Woltjen et al., “PiggyBac Transposition ReprogramsFibroblasts to Induced Pluripotent Stem Cells,” Nature 458: 766-770(2009), and Yusa et al., “Generation of Transgene-Free InducedPluripotent Mouse Stem Cells by the PiggyBac Transposon,” Nat. Methods6: 363-369 (2009), which are hereby incorporated by reference in theirentirety. Suitable methods for iPSC generation also include methodsinvolving the direct delivery of reprogramming factors as recombinantproteins (Zhou et al., “Generation of Induced Pluripotent Stem CellsUsing Recombinant Proteins,” Cell Stem Cell 4: 381-384 (2009), which ishereby incorporated by reference in its entirety) or as whole-cellextracts isolated from ESCs (Cho et al., “Induction of Pluripotent StemCells from Adult Somatic Cells by Protein-Based Reprogramming withoutGenetic Manipulation,” Blood 116: 386-395 (2010), which is herebyincorporated by reference in its entirety).

The methods of iPSC generation described above can be modified toinclude small molecules that enhance reprogramming efficiency or evensubstitute for a reprogramming factor. These small molecules include,without limitation, epigenetic modulators such as the DNAmethyltransferase inhibitor 5′-azacytidine, the histone deacetylaseinhibitor VPA, and the G9a histone methyltransferase inhibitor BIX-01294together with BayK8644, an L-type calcium channel agonist. Other smallmolecule reprogramming factors include those that target signaltransduction pathways, such as TGF-β inhibitors and kinase inhibitors(e.g., kenpaullone) (see review by Sommer and Mostoslaysky,“Experimental Approaches for the Generation of Induced Pluripotent StemCells,” Stem Cell Res. Ther. 1:26 doi:10.1186/scrt26 (Aug. 10, 2010),which is hereby incorporated by reference in its entirety).

Suitable iPSCs derived from adult fibroblasts can be obtained followingthe procedure described in Streckfuss-Bomeke et al., “Comparative Studyof Human-Induced Pluripotent Stem Cells Derived from Bone Marrow Cells,Hair Keratinocytes, and Skin Fibroblasts,” Eur. Heart J. doi:10.1093/eurheartj/ehs203 (2012), which is hereby incorporated byreference in its entirety). iPSCs derived from umbilical cord bloodcells can be obtained as described in Cai et al., “Generation of HumanInduced Pluripotent Stem Cells from Umbilical Cord Matrix and AmnioticMembrane Mesenchymal Cells,” J. Biol. Chem. 285(15): 112227-11234 (2110)and Giorgetti et al., “Generation of Induced Pluripotent Stem Cells fromHuman Cord Blood Cells with only Two Factors: Oct4 and Sox2,” NatureProtocols, 5(4):811-820 (2010), which are hereby incorporated byreference in their entirety. iPSCs derived from bone marrow cells can beobtained using methods described in Streckfuss-Bomeke et al.,“Comparative Study of Human-Induced Pluripotent Stem Cells Derived fromBone Marrow Cells, Hair Keratinocytes, and Skin Fibroblasts,” Eur. HeartJ. doi: 10.1093/eurheartj/ehs203 (Jul. 12, 2012), and Hu et al.,“Efficient Generation of Transgene-Free Induced Pluripotent Stem Cellsfrom Normal and Neoplastic Bone Marrow and Cord Blood MononuclearCells,” Blood doi: 10.1182/blood-2010-07-298331 (Feb. 4, 2011) which arehereby incorporated by reference in their entirety). iPSCs derived fromperipheral blood can be obtained following the methods described inSommer et al., “Generation of Human Induced Pluripotent Stem Cells fromPeripheral Blood using the STEMCCA Lentiviral Vector,” J. Vis. Exp. 68:e4327 doi:10.3791/4327 (2012), which is hereby incorporated by referencein its entirety. iPS cells contemplated for use in the methods of thepresent invention are not limited to those described in the abovereferences, but rather includes cells prepared by any method as long asthe cells have been artificially induced from cells other thanpluripotent stem cells.

As described herein and shown in FIG. 1A, oligodendrocyte progenitorcells can be derived from iPSCs using a protocol that directs the iPSCsthrough serial stages of neural and glial progenitor celldifferentiation. Each stage of lineage restriction is characterized andidentified by the expression of certain cell proteins.

With reference to FIG. 1A, Stage 1 of the process involves culturingiPSCs under conditions effective to induce embryoid body formation. Asdescribed herein, iPSCs may be maintained in co-culture with othercells, such as embryonic fibroblasts, in an embryonic stem cell (ESC)media (e.g., DMEM/F12 containing a suitable serum replacement and bFGF).The iPSCs are passaged before reaching 100% confluence, e.g., 80%confluence, when colonies are approximately 250-300 μm in diameter. Thepluripotential state of the cells can be readily assessed using markersto SSEA4, TRA-1-60, OCT-4, NANOG, and/or SOX2.

To generate embryoid bodies (EBs) (Stage 2), which are complexthree-dimensional cell aggregates of pluripotent stem cells, iPSCcultures are dissociated once they achieved ˜80% confluence with colonydiameters at or around 250-300 μm. The EBs are initially cultured insuspension in ESC media without bFGF, and then switched to neuralinduction medium supplemented with bFGF and heparin. To induceneuroepithelial differentiation (Stage 3), EBs are plated and culturedin neural induction medium supplemented with bFGF, heparin, laminin,then switched to neural induction media supplemented with retinoic acid.Neuroepithelial differentiation is assessed by the co-expression of PAX6and SOX1, which characterize central neural stem and progenitor cells.

To induce pre-oligodendrocyte progenitor cell (“pre-OPCs”)differentiation, neuroepithelial cell colonies are cultured in thepresence of additional factors including retinoic acid, B27 supplement,and a sonic hedgehog (shh) agonist (e.g., purmophamine). The appearanceof pre-OPC colonies can be assessed by the presence of OLIG2 and/orNKX2.2 expression. While both OLIG2 and NKX2.2 are expressed by centraloligodendrocyte progenitor cells, NKX2.2 is a more specific indicator ofoligodendroglial differentiation. Accordingly, an earlypre-oligodendrocyte progenitor cell stage is marked by OLIG⁺/NKX2.2⁻cell colonies. OLIG⁺/NKX2.2⁻ early pre-OPCs are differentiated intolater-stage OLIG⁺/NKX2.2⁺ pre-OPCs by replacing retinoic acid with bFGF.At the end of Stage 5, a significant percentage of the cells arepre-OPCs as indicated by OLIG2⁺/NKX2.2⁺ expression profile.

Pre-OPCs are further differentiated into bipotential oligodendrocyteprogenitor cells by culture in glial induction media supplemented withgrowth factors such as triiodothyronine (T3), neurotrophin 3 (NT3),insulin growth factor (IGF-1), and platelet-derived growth factor-AA(PDGF-AA) (Stage 6). These culture conditions can be extended for 3-4months or longer to maximize the production of myelinogenicoligodendrocyte progenitor cells. Cell preparations suitable fortherapeutic transplant are identified as containingOLIG2⁺/NKX2.2⁺/SOX10⁺/PDGFRα⁺ oligodendrocyte progenitor cells. In vitroterminal differentiation of oligodendrocyte progenitor cells intooligodendrocytes, identified by O4⁺ and myelin basic protein (MBP),arose in further culture with a reduction of glial mitogens.

In some embodiments of the present invention, it may be preferable toenrich for oligodendrocyte progenitor cells within a cell preparationproduced using the methods described herein. Accordingly, in oneembodiment, selection of CD140a/PDGFRα positive cells is employed toproduce a purified or enriched preparation of oligodendrocyte progenitorcells. In another embodiment of the present invention, selection of CD9positive cells is employed to produce a purified or enriched preparationof oligodendrocyte progenitor cells. In yet another embodiment, bothCD140a/PDGFRα and CD9 positive cell selection is employed to produce apurified or enriched preparation of oligodendrocyte progenitor cells.

Selection for a PDGFRα marker and/or a CD9 marker can be carried outserially or sequentially and can be performed using conventional methodsknown in the art such as immunopanning. The selection methods optionallyinvolve the use of fluorescence sorting (FACS), magnetic sorting (MACS)or any other methods that allow a rapid, efficient cell sorting.Examples of methods for cell sorting are taught for example in U.S. Pat.No. 6,692,957, which is hereby incorporated by reference in itsentirety.

Generally, cell sorting methods use a detectable moiety. Detectablemoieties include any suitable direct or indirect label, including, butnot limited to, enzymes, fluorophores, biotin, chromophores,radioisotopes, colored beads, electrochemical, chemical-modifying orchemiluminescent moieties. Common fluorescent moieties includefluorescein, cyanine dyes, coumarins, phycoerythrin, phycobiliproteins,dansyl chloride, Texas Red, and lanthanide complexes or derivativesthereof. Magnetic cell sorting may be used.

When cell sorting is performed, the marker can be an ectodomain and cellpermeabilization or membrane disruption techniques are not used. By wayof example, the PDGFRα marker selection step is optionally performedusing an antibody or other binding moiety that binds an ectodomain ofthe PDGFRα (e.g. CD140a). Suitable antibodies include, but are notlimited to, monoclonal and polyclonal antibodies, chimeric antibodies,antibody fragments (e.g., F(ab′)2, Fab′, Fab fragments) capable ofbinding the selected marker, and single chain antibodies. Other bindingmoieties include marker ligands, cofactors, and the like thatspecifically bind to the marker. Thus, in the case of a marker that is areceptor, a receptor ligand or binding portion thereof can be used as adetectable moiety. Antibodies and other binding moieties arecommercially available or can be made using techniques available to askilled artisan.

One of skill in the art readily appreciates how to select for or againsta specific marker. Thus, by way of example, a population of cells sortedfor a particular marker includes identifying cells that are positive forthat particular marker and retaining those cells for further use orfurther selection steps. A population of cells sorted against a specificmarker includes identifying cells that are positive for that particularmarker and excluding those cells for further use or further selectionsteps.

Another aspect of the present invention is directed to a method oftreating a subject having a condition mediated by a loss of myelin or aloss of oligodendrocytes. This method involves administering to thesubject any one of the oligodendrocyte progenitor cell preparations ofthe present invention under conditions effective to treat the condition.In accordance with this aspect of the present invention, any of cellpreparations described herein are suitable for therapeutic treatment.

Conditions mediated by a loss of myelin or a loss of oligodendrocytesthat can be treated in accordance with the methods of the presentinvention include hypomyelination disorders and demyelinating disorders.In one embodiment of the present invention, the condition is anautoimmune demyelination condition, such as e.g., multiple sclerosis,neuromyelitis optica, transverse myelitis, and optic neuritis. Inanother embodiment of the present invention, the myelin-related disorderis a vascular leukoencephalopathy, such as e.g., subcortical stroke,diabetic leukoencephalopathy, hypertensive leukoencephalopathy,age-related white matter disease, and spinal cord injury. In anotherembodiment of the present invention, the myelin-related condition is aradiation induced demyelination condition. In another embodiment of thepresent invention, the myelin-related disorder is a pediatricleukodystrophy, such as e.g., Pelizaeus-Merzbacher Disease, Tay-SachDisease, Sandhoff's gangliosidoses, Krabbe's disease, metachromaticleukodystrophy, mucopolysaccharidoses, Niemann-Pick A disease,adrenoleukodystrophy, Canavan's disease, Vanishing White Matter Disease,and Alexander Disease. In yet another embodiment of the presentinvention, the myelin-related condition is periventricular leukomalaciaor cerebral palsy.

The number of oligodendrocyte progenitor cells administered to thesubject can range from about 10²-10⁸ cells at each transplantation(e.g., injection site), depending on the size and species of therecipient, and the volume of tissue requiring myelin production orreplacement. Single transplantation (e.g., injection) doses can spanranges of 10³-10⁵, 10⁴-10⁷, and 10⁵-10⁸ cells, or any amount in totalfor a transplant recipient patient.

Delivery of the cells to the subject can include either a single step ora multiple step injection directly into the nervous system.Specifically, the cells can be delivered directly to one or more sitesof the brain, the brain stem, the spinal cord, and/or any combinationthereof. For localized disorders such as demyelination of the opticnerve, a single injection can be used. Although the oligodendrocyteprecursor cells of the present invention disperse widely within atransplant recipient's brain, for widespread demyelinating orhypomyelination disorders, multiple injections sites can be performed tooptimize treatment. Injection is optionally directed into areas of thecentral nervous system such as white matter tracts like the corpuscallosum (e.g., into the anterior and posterior anlagen), dorsalcolumns, cerebellar peduncles, cerebral peduncles via intraventricular,intracallosal, or intraparenchymal injections. Such injections can bemade unilaterally or bilaterally using precise localization methods suchas stereotaxic surgery, optionally with accompanying imaging methods(e.g., high resolution MRI imaging). One of skill in the art recognizesthat brain regions vary across species; however, one of skill in the artalso recognizes comparable brain regions across mammalian species.

The cellular transplants are optionally injected as dissociated cellsbut can also be provided by local placement of non-dissociated cells. Ineither case, the cellular transplants optionally comprise an acceptablesolution. Such acceptable solutions include solutions that avoidundesirable biological activities and contamination. Suitable solutionsinclude an appropriate amount of a pharmaceutically-acceptable salt torender the formulation isotonic. Examples of thepharmaceutically-acceptable solutions include, but are not limited to,saline, Ringer's solution, dextrose solution, and culture media. The pHof the solution is preferably from about 5 to about 8, and morepreferably from about 7 to about 7.5.

The injection of the dissociated cellular transplant can be a streaminginjection made across the entry path, the exit path, or both the entryand exit paths of the injection device (e.g., a cannula, a needle, or atube). Automation can be used to provide a uniform entry and exit speedand an injection speed and volume.

Optionally a multifocal delivery strategy can be used, for example asdescribed in the examples. Such a multifocal delivery strategy isdesigned to achieve widespread, dense, whole neuraxis donor cellengraftment throughout the recipient central nervous system. Injectionsites can be chosen to permit contiguous infiltration of migrating donorcells into one or more of the major brain areas, brainstem, and spinalcord white matter tracts, without hindrance (or with limited hindrance)from intervening gray matter structures. For example, injection sitesoptionally include four locations in the forebrain subcortex,specifically into the anterior and posterior anlagen of the corpuscallosum bilaterally, and into a fifth location in the cerebellarpeduncle dorsally.

Optionally, the methods of treatment provided herein further compriseassessing remyelination directly or indirectly. For example, imaginingtechnique, conduction velocities, or symptomatic improvement areoptionally tested subsequent to engraftment.

An advantage of the oligodendrocyte progenitor cells of the presentinvention is the ability to obtain patient specific preparation forautologous cell therapy (i.e., the cell preparation is derived from thesubject being treated). Autologous oligodendrocyte progenitor cells canbe derived, for example, from the somatic skin cells, umbilical chordblood, peripheral blood, or bone marrow of the subject being treated.Oligodendrocyte progenitor cells derived from allogeneic and/orxenogeneic cell sources are also suitable for use in the methods of thepresent invention.

Examples

The following examples are provided to illustrate embodiments of thepresent invention but they are by no means intended to limit its scope

Material and Methods for Examples 1-9

hESC and hiPSC Culture.

Four distinct lines of pluripotent cells were used in this study. Theseincluded hESCs (WA09/H9; WiCell, Madison, Wis., USA) and hiPSCs of bothkeratinocyte (K04; K. Hochedlinger) and fibroblast origin (C14 and C27hiPSCs; L. Studer). The experiments described were approved by theUniversity of Rochester Embryonic Stem Cell Research Oversightcommittee.

OPC Production.

OPCs were induced from hESCs and iPSCs using modifications of publishedprotocols (Hu et al., “Human Oligodendrocytes From Embryonic Stem Cells:Conserved SHH Signaling Networks and Divergent FGF Effects,” Development136:1443-1452 (2009); Hu et al., “Differentiation of HumanOligodendrocytes From Pluripotent Stem Cells,” Nat. Protoc. 4:1614-1622(2009b); Izrael et al., “Human Oligodendrocytes Derived From EmbryonicStem Cells: Effect of Noggin on Phenotypic Differentiation in Vitro andon Myelination in Vivo,” Mol. Cell. Neurosci. 34:310-323 (2007), whichare hereby incorporated by reference in their entirety), as schematizedin FIG. 1 and described in detail below.

Directed Astrocytic or Oligodendrocytic Maturation.

hiPSC- or hESC-derived gliogenic spheres at 120-170 DIV were cultured insuspension in GIM supplemented with platelet-derived growth factor-AA(PDGF-AA; 10 ng/ml), insulin growth factor-1 (IGF-1; 10 ng/ml) andNeurotrophin-3 (NT3; 10 ng/ml). To differentiate these OPCs into matureoligodendrocytes (OLs) or astrocytes, the spheres were dissected intosmall cell clusters (around 50-100 mm in diameter) mechanically with aSharpoint blade (Surgimed-MLB). The dissected OPC clusters were platedonto polyornithine/laminin-coated 12-well plates and cultured in GIM for1-2 weeks. For induction of astrocytes, the OPC clusters were culturedeither in GIM supplemented with PDGF-AA (10 ng/ml), IGF-1 (10 ng/ml),and NT3 (10 ng/ml) or in GIM supplemented with 10% fetal bovine serum(FBS; HyClone) for 1-2 weeks. For directing the maturation of OLs, thecultures were switched to half GIM supplemented with PDGF-AA (5 ng/ml),IGF-1 (5 ng/ml), and NT3 (5 ng/ml) plus half neurobasal (NB) media(Invitrogen) supplemented with B27 (1×) and brain-derived neurotrophicfactor (BDNF; 10 ng/ml) and grown for 2-4 weeks. The mature astrocyteswere recognized with immunostaining of anti-GFAP or anti-CD44.Oligodendroglia were identified using 04 and MBP antibodies.

Isolation of Human Fetal Neuronal Progenitor Cells for Coculture.

Human fetal forebrain tissue was obtained from second-trimester abortedfetuses of 20 weeks g.a. Tissues were obtained as de-identified tissue,as approved by the Research Subjects Review Board of the University ofRochester Medical Center. The tissue samples were washed 2-3 times withsterile Hank's balanced salt solution with Ca²⁺/Mg²⁺ (HBSS^(+/+)).Cortical plate tissue was separated from the ventricularzone/subventricular zone, then dissociated with papain (WorthingtonBiochemical) as described (Keyoung et al., “High-Yield Selection andExtraction of Two Promoter-Defined Phenotypes of Neural Stem Cells Fromthe Fetal Human Brain,” Nat. Biotechnol. 19:843-850 (2001); Wang et al.,“Prospective Identification, Isolation, and Profiling of aTelomerase-Expressing Subpopulation of Human Neural Stem Cells, Usingsox2 Enhancer-Directed Fluorescence-Activated Cell Sorting,” J.Neurosci. 30:14635-14648 (2010), which are hereby incorporated byreference in their entirety). The cells were resuspended at 2-4×10⁶cells/ml in Dulbecco's modified Eagle's medium (DMEM)/F12 supplementedwith N-2 supplement (Life Technologies) and basic fibroblast growthfactor (bFGF; 20 ng/ml) and plated in suspension culture dishes. A daylater, the cells were recovered and neurons isolated bymagnetic-activated cell sorting (Windrem et al., “Neonatal ChimerizationWith Human Glial Progenitor Cells Can Both Remyelinate and Rescue theOtherwise Lethally Hypomyelinated Shiverer Mouse,” Cell Stem Cell2:553-565 (2008), which is hereby incorporated by reference in itsentirety). In brief, the recovered neural progenitor cells wereincubated with PSA-NCAM (Chemicon) at 1:100 for 30 min, then washed andlabeled with rat anti-mouse immunoglobulin M microbeads (MiltenyiBiotech). The bound PSA-NCAM⁺ neurons were eluted, spun, washed withDMEM/F12, and then cultured in DMEM/F12 with N2, 0.5% PD-FBS, and bFGF(20 ng/ml) for 4-6 days. For coculture with hiPSC OPCs, the fetalcortical neurons were dissociated into single cells and then plated ontoeither poly-L-ornithine/laminin-coated 24-well plates orpoly-L-ornithine/fibronectincoated coverslips (50,000-100,000 cells perwell or coverslip). The replated neurons were then switched to NB mediawith B27 (1×) and BDNF (10 ng/ml; Invitrogen) for an additional 6-10days prior to coculture.

Coculture of hiPSC-Derived OPCs with Human Fetal Cortical Neurons InVitro.

Gliogenic OPC spheres derived from either K04 or C27 hiPSCs were inducedup to 130 DIV prior to coculture. These were dissected into small piecesof <1 mm³ and cultured for 2-3 weeks to allow the OPCs to expand as amonolayer surrounding the core clusters. The OPC clusters and theirmonolayer surrounds were then recollected with cold HBSS^(−/−) from theculture dishes and manually dissected into smaller fragments of 100-200μm in diameter. Small aliquots were fully dissociated into single cellswith Accutase (Chemicon) for 5 min at room temperature, then assessed byhemocytometry. For coculture, the hiPSC OPCs were then seeded at 200,000cells/ml, either with or without human cortical neurons, and cultured ina 1:1 mixture of NB/B27/BDNF and GIM/NT3/IGF-1/PDGF-AA media. Thecultures of cortical neurons alone, hiP SC OPCs alone, or bothpopulations together were allowed to grow 2-4 additional weeks beforefixation and immunolabeling for O4, MBP, GFAP, and βIII-tubulin.

RNA Extraction and RT-PCR.

Total RNA was extracted from undifferentiated hESCs and hiPSCs, or hESC-and hiPSC-derived OPCs, using RNeasy mini kit (QIAGEN). The first ofstrand of complementary DNA was transcribed using the TaqMan ReverseTranscription kit (Roche #N808-0234). The primer sequences are providedin Table 1 below. The relative abundance of transcript expression ofmRNAs was measured with the ABI PRISM 7000 system. The resultantexpression data were normalized to the expression level ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Statisticalanalysis was performed on transformed data. The means and SEM werecalculated following a paired t test.

TABLE 1 Primers of human genes used for qRT-PCR Target Accession GeneForward primer Reverse primer number c-MYC CGTCTCCACACATCAGCACAATCTTGGCAGCAGGATAGTCCTT NM_002467.4 (SEQ ID NO: 1) (SEQ ID NO: 2) GAPDHCCACCCATGGCAAATTCC TGGGATTTCCATTGATGACA NM_002046.3 (SEQ ID NO: 3)(SEQ ID NO: 4) GFAP CATCGAGATCGCCACCTACA TCTGCACGGGAATGGTGATNM_001131019.1 (SEQ ID NO: 5) (SEQ ID NO: 6) hTERT TGCGGCCGATTGTGAACCCTCTTTTCTCTGCGGAACG NM_001193376.1 (SEQ ID NO: 7) (SEQ ID NO: 8) KLF4ACCAGGCACTACCGTAAACACA GGTCCGACCTGGAAAATGCT NM_004235.4 (SEQ ID NO: 9)(SEQ ID NO: 10) NANOG CCAAAGGCAAACAACCCACTT TCTTGACCGGGACCTTGTCTNM_024865.2 (SEQ ID NO: 11) (SEQ ID NO: 12) NKX2.2 GGCGGGCATTCCCTTTTCGAGCTGTACTGGGCGTTGT NM_002509.3 (SEQ ID NO: 13) (SEQ ID NO: 14) OCT4TGGTCCGAGTGTGGTTCTGTAA TGTGCATAGTCGCTGCTTGAT NM_001173531.1(SEQ ID NO: 15) (SEQ ID NO: 16) OLIG2 GGCGCGCAACTACATCCTCGCTCACCAGTCGCTTCAT NM_005806.2 (SEQ ID NO: 17) (SEQ ID NO: 18) PAX6TCGGGCACCACTTCAACAG TCCGGGAACTTGAACTGGAA NM_000280.3 (SEQ ID NO: 19)(SEQ ID NO: 20) PDGFR CCTTGGTGGCACCCCTTAC TCCGGTACCCACTCTTGATCTTNM_006206 (SEQ ID NO: 21) (SEQ ID NO: 22) SOX2 TGCGAGCGCTGCACATGCAGCGTGTACTTATCCTTCTTCA NM_003106.2 (SEQ ID NO: 23) (SEQ ID NO: 24)SOX10 CCACGAGGTAATGTCCAACATG CATTGGGCGGCAGGTACT NM_006941.3(SEQ ID NO: 25) (SEQ ID NO: 26)

Neonatal Xenograft into Shiverer Mice.

Homozygous shiverer mice (The Jackson Laboratory, Bar Harbor, Me., USA)were crossed with homozygous rag2-null immunodeficient mice (Shinkai etal., “RAG-2-Deficient Mice Lack Mature Lymphocytes Owing to Inability toInitiate V(D)J Rearrangement,” Cell 68:855-867 (1992), which is herebyincorporated by reference in its entirety) on the C3H background(Taconic, Germantown, N.Y., USA) for generation of shi/shi x rag2^(−/−)myelin-deficient, immunodeficient mice. The hiPSC-derived OPCs wereprepared for transplantation as described for in vitro coculture.Neonatal pups were either transplanted bilaterally in the corpuscallosum with a total of 100,000 cells, as described in Windrem et al.,“Fetal and Adult Human Oligodendrocyte Progenitor Cell IsolatesMyelinate the Congenitally Dysmyelinated Brain,” Nat. Med. 10:93-97(2004), which is hereby incorporated by reference in its entirety, orwith 300,000 cells, using the procedure described in Windrem et al.,“Neonatal Chimerization With Human Glial Progenitor Cells Can BothRemyelinate and Rescue the Otherwise Lethally Hypomyelinated ShivererMouse,” Cell Stem Cell 2:553-565 (2008), which is hereby incorporated byreference in its entirety. At 3 months of age, transplanted mice wereanesthetized with pentobarbital, then perfusion fixed with coldHBSS^(+/+) followed by 4% paraformaldehyde. All procedures were approvedby the University Committee on Animal Resources. Brains were extractedand postfixed for 2 hr in cold paraformaldehyde. Brains processed forelectron microscopy were perfused in 4% paraformaldehyde and 0.25%glutaraldehyde.

Immunohistochemistry of Tissue Sections.

Human cells were identified with mouse anti-hNA, clone 235-1 (MAB1281 at1:800; Millipore, Billerica, Mass., USA). Phenotypes were identifiedwith human-specific NG2 (MAB2029 at 1:200; Millipore), rat anti-MBP(Ab7349 at 1:25), rabbit anti-OLIG2 (Ab33427 at 1:1000; Abcam,Cambridge, Mass., USA), human-specific mouse anti-GFAP (SMI-21 at1:500), mouse anti-NF (SMI-311 and SMI-312 at 1:1,000; Covance,Princeton, N.J., USA), and rabbit anti-Ki67 (RM-9106 at 1:200;Thermo-Fisher, Freemont, Calif., USA). Alexa Fluor secondary antibodies,including goat anti-mouse, -rat, and -rabbit antibodies conjugated to488, 568, 594, and 647 nm fluorophores, were used at 1:400 (Invitrogen,Carlsbad, Calif., USA). PAX6, NKX2.2, OCT4, NANOG, and SOX2 antibodieswere employed using the same conditions as in vitro.

Myelinated Axon Counts.

Regions of dense engraftment with human cells were selected for NF andMBP staining; a 1 μm stack of ten superimposed optical slices taken at0.1 μm intervals (Olympus FluoView 300) was made for each of threefields of view in the corpus callosum. Three parallel, equidistant lineswere laid over the images perpendicular to the axons. Axons were scoredat intersections with the lines as either myelinated (closely apposed toMBP on both sides) or unmyelinated.

Mapping of Human Cell Engraftment.

The positions of all anti-human nuclei⁺ cells were mapped on 20 μmcoronal sections at 160 μm intervals from −3.2 to 1.2 bregmaanterior-posterior.

Cell Counting.

Three unilateral, equally spaced samples of corpus callosum, from −0.4to 1.2 bregma, were counted for cells expressing hNA together witheither MBP, hGFAP, OLIG2, or Ki67. White matter was also assessed forthe presence of any hNA⁺ cells coexpressing HuC/HuD, OCT4, or NANOG. Alldata are provided as means±SEM.

Electron Microscopy.

Samples of human iPSC-derived glial chimeric white matter were takenfrom mice killed at 22-40 weeks of age, perfused with half-strengthKarnovsky's fixative, then processed for ultrastructural analysis ofmyelin morphology and quality using previously described techniques(Windrem et al., “Neonatal Chimerization With Human Glial ProgenitorCells Can Both Remyelinate and Rescue the Otherwise LethallyHypomyelinated Shiverer Mouse,” Cell Stem Cell 2:553-565 (2008), whichis hereby incorporated by reference in its entirety).

Cell Preparation.

Stage 1 Both hESCs and hiPSCs were cultured on irradiated mouseembryonic fibroblast (MEF) cells, and fed daily with hESC medium,consisting of DMEM/F12 containing with 20% KO-serum replacement,supplemented with bFGF (4 ng/ml, Invitrogen). Both hESC and hiPSCs werepassaged when they reached 80% confluence in colonies of 250-300 μmdiameter, typically every 3-4 days for WA09/H9 and every 4 (K04 cells)or 7 days (C14 and C27) for hiPSCs. The undifferentiated stem cells werevalidated immunocytochemically for their expression of human pluripotentstem cell markers, that included SSEA4, TRA-1-60, OCT4, NANOG, and SOX2(FIG. 2). Of note, both OCT4 and SOX2 were also utilized asreprogramming factors in the generation of the hiPSCs, the generation ofwhich have been previously described. Cells were passaged by incubationin collagenase type IV (1 mg/ml, Invitrogen) for 5-10 min, followed bygentle scraping from the culture dish, after which they were triturated5 times through a polished glass pipette and then spun, washed andresuspended twice. The cells were then split 1:3-1:4 onto 6-well platespre-coated with irradiated MEF cells.

Stage 2

To generate embryonic bodies (EB), hESC or hiPSC cultures weredissociated using Dispase (0.5 mg/ml, Invitrogen) at 37° C. for 5-10min, once they achieved 80% confluence with colony diameters of 250-300m, in the absence of evident differentiation. These criteria provedimportant, as it was noted that the quality of hESC and hiPSC culturesat the stage 1-2 transition critically affected that of their derivedEBs, as well as their subsequent differentiation into OPCs. The EBs werecultured in suspension in tissue culture flasks (Nunc EasYFlasks, ThermoScientific) in ESC medium without bFGF for 5 days; then switched toneural induction medium (NIM; DMEM/F12 supplemented with non-essentialamino acids and N2) supplemented with bFGF (20 ng/ml, Sigma) and heparin(2 μg/ml, Sigma), for either 2 days (WA9/H9 hES) or 7 days (K04, C14 andC27 hiPSCs). Thereafter, the EBs were plated onto laminin/poly-ornithinecoated 6-well plates and cultured in NIM supplemented with bFGF, heparinand laminin (10 μg/ml) for 3 additional days; the medium was thenswitched to NIM supplemented with retinoic acid (RA, 100 nM, Sigma), for4 days.

Stage 3

The neuroepithelial differentiation efficiency at this point, the end ofstage 3, was assessed by immunolabeling for PAX6 and SOX1; co-expressionof these markers characterizes central neural stem and progenitor cells.The yields of neuroepithelial colonies, defined as (PAX6⁺/SOX1⁺)/totalrosette-like colonies, were 52.2±7.5%, 78.4±4.7%, and 76.0±7.0% fromK04, C14 and C27 cultures, respectively (N=3-6 scored cultures/line).The efficiencies of neuroepithelial colony production from C14 and C27hiPSCs were similar to those for WA09/H9 (75.4±8.8%) (FIGS. 3A and 4A).

Stage 4

On day 14 (for H9) or day 19 (for K04 and C27) (end of stage 3, 4 daysafter addition of RA), purmorphamine (1 μM, Calbiochem), a sonichedgehog (shh) agonist, and B27 (Invitrogen) were added to the media.The cultured NE colonies were detached mechanically 9 days later, ateither 23 DIV (WA9/H9 hES) or 28 DIV (K04, C14 and C27 hiPSC), and thencultured in suspension in 6-well Ultralow cluster plates (FIG. 4B).

Stage 5

One day after plating into Ultralow cluster plates, the medium wasreplaced with NIM supplemented with bFGF (10 ng/ml), in addition topurmophamine and B27. At that point the phenotypic composition ofaliquots was assessed by staining of OLIG2 and/or NKX2.2, to ascertainthe appearance of pre-OPC colonies following RA treatment. Both OLIG2and NKX2.2 are expressed by central OPCs, though NKX2.2 is the morespecific indicator of oligodendroglial differentiation (see citations16-17). At this early pre-OPC stage, the percentage of OLIG2-expressingcolonies was higher than that of NKX2.2⁺ colonies, reflecting theearlier appearance of OLIG2 (FIGS. 3A and 4C). In contrast, by the endof stage 5 (35 DIV for WA09/H9 or 40 DIV for K04, C14 and C27), underthe effect of bFGF without RA, more NKX2.2⁺ colonies appeared,concurrent with the peak of OLIG2 expression. By the end of this stage,the percentage of OLIG2⁺/NKX2.2⁺ co-expressing colonies was similaramong all four lines in this study (FIG. 3A).

Stage 6

To initiate stage 6 (day 35 for WA09/H9 or day 40 for K04, C14 and C27),the OLIG2/NKX2.2-defined pre-OPCs in stage 5 suspension culture wereswitched to glial induction media (GIM; DMEM/F12, N1, B27, T3 at 60ng/ml, biotin at 100 ng/ml, dibutyryl-cAMP at 1 μM; all from Sigma)supplemented with PDGF AA (10 ng/ml), IGF-1 (10 ng/ml), and NT3 (10ng/ml). During this long period of OPC suspension culture, ⅔ of themedia volume was changed every 3 days. The resultant stage 6 gliosphereswere prevented from aggregating by gentle trituration through P1000pipette tips during media changes.

Beginning at 95 DIV, the efficiency of hOPC differentiation, as definedby A2B5, CD140a and CD140a/CD9 co-expression, was assessed both in vitroand in vivo, using ICC, qRT-PCR, and xenograft into neonatal shiverermice at serial time points. Gliospheres were capable of yielding bothmature oligodendrocytes and myelinogenic OPCs as of 120 DIV. Between120-200 DIV, the incidence of OPC-bearing colonies rose steadily, suchthat the proportion of OLIG2 and NKX2.2 co-expressing colonies of OPCsfrom K04, C14 and C27 were 73.8±8.7%, 78.9±6.1% and 79.5±8.5%,respectively. Interestingly, the efficiency of hOPC production by hiPSCcells was consistently higher than that exhibited by WA09/H9 cells(45.4±20.3%) (FIG. 3A).

Late Stage 6/Pre-Transplant

By this point (late stage 6), the newly produced hOPCs also expressedother OPC markers, such as CD140a/PDGFRα and SOX10, which typicallyco-expressed OLG2 or NKX2.2 (FIGS. 4D-4H). At this stage the percentagesof the OLG2⁺, NXK2.2⁺ or SOX10⁺, among all DAPI-identified cells, were61.9±10.3%, 63.4±7.3%, and 84.6±7.0% among hiPCS/K04-derived OPCs, whilethe corresponding proportion of NKX2.2/SOX10 co-expressing OPCs was60.6±4.4% (FIG. 4I). To further validate the efficiency of OPCdifferentiation, RT-PCR for OLIG2, NKX2.2 and GFAP mRNA was performed,and all genes were substantially upregulated in stage 6 OPCs, as weretheir corresponding protein products (FIGS. 11C-D and 3C).

Flow Cytometric Protocols and Analysis.

Flow cytometry of hESC- or hiPSC-derived OPCs was performed on aFACSAria IIIU (Becton Dickinson, San Jose, Calif.). Cells were gentlyscraped from the culture dishes and then treated with Accutase(Chemicon) at 37° C. for 5 minutes with gentle shaking. The samples werethen triturated with a narrow glass Pasteur pipette until a single cellsuspension was obtained. The cells were then spun and resuspended inMiltenyi Washing Buffer (MWB) at 1×10⁶ cells/ml. The primary antibodies,directly conjugated antibodies or their corresponding isotype controlswere added to the cells at the concentrations listed below, thenincubated on ice for 15 minutes. 5 ml of MWB was added and the cellswere spun down. For the non-conjugated antibodies, the pelleted cellswere resuspended in MWB to 1×10⁶ cells/ml and the appropriate secondaryantibody, Alexa-488 conjugated goat anti-mouse IgM, was added at 1:500dilution. The samples were incubated on ice for 15 minutes and thenwashed with 5 ml of MWB for 10 minutes. All samples were thenresuspended in Phenol Red-free DMEM/F-12 to a concentration of 1-1.5×10⁶cells/ml, then passed through a 40-μm cell strainer (Becton Dickinson,BD). DAPI was added at 1 g/ml. The cells were analyzed by forward andside scatter, for PE fluorescence through a 582±15 nm band-pass filter,for Alexa Fluor 488/FITC fluorescence through a 530±30 nm band-pass, forPERCP-Cy5.5 through a 695±40 nm band-pass, and for DAPI fluorescencethrough a 450±50 nm band-pass. Unstained cells were used to set thebackground fluorescence; a false positive rate of 0.5% was accepted. Theantibodies used were mouse IgM isotype control (Chemicon, PP50), mouseanti-A2B5 (IgM, Chemicon, MAB312), mouse anti-04 (IgM, Chemicon,MAB345), PE mouse IgG_(2a), x isotype control (BD, 555574), PE mouseanti-human CD140a (IgG_(2a), BD, 556002), PERCP-Cy5.5 mouse IgG₁ isotypecontrol (BD, 347212) and PERCP-Cy5.5 mouse anti-human CD9 (IgG₁, BD,341649).

In Vitro Immunocytochemistry.

Pluripotent hESC or hiPSCs raised on irradiated MEF cells were culturedfor 3 to 4 days prior to fixation with 4% paraformaldehyde. Similarly,the differentiated neurogenic or gliogenic clusters were plated ontopoly-ornithine and laminin coated 24-well plate and cultured for 3 daysbefore being fixed with 4% paraformaldehyde. The gliogenic spherescontaining hOPCs at later stages were dissected into small fragments andplated onto poly-ornithine/laminin coated 24-well plates, and culturedfor 2-4 weeks before being fixed, depending on the experiment. Fixationwas performed with 4% paraformaldehyde for 5 min at room temperaturefollowed by 3 washes with PBS. Immunolabeled cells were incubated withprimary antibodies overnight at 4° C. and with secondary antibodies for0.5 h at 25° C. Primary antibodies included: mouse anti-OCT4, mouseanti-SSEA4, mouse anti-TRA-60 (all were used in 1:100 dilution and werefrom Chemicon); rabbit anti-NANOG (1:500, Abcam); rabbit anti-PAX6(1:400, Covance); goat anti-OLIG2 (1:200, R&D); mouse anti-NKX2.2(1:100, DSHB); rabbit anti-PDGFR (1:400, Santa Cruz Biotechnology);rabbit anti-SOX10 (1:400, Advanced Bioscience Resources); goat anti-SOX1(1:100, R&D Systems); goat anti-SOX2 (1:1000, R&D Systems mouseanti-GFAP (1:400, Covance); rabbit anti-GFAP (1:1000, Chemicon); NESTIN(1:1000, Millipore Bioscience Research Reagents); mouse anti-III-tubulin(1:1000, Covance); oligodendrocytic sulfatide, as recognized by MAb O4(1:100, Millipore Bioscience); and rat anti-MBP (1:25, Abcam).

Example 1—Human iPSCs can be Efficiently Directed to Glial ProgenitorCell Fate

Four different iPSC lines from three different sources were used forthis study; these included WA09/H9 hESCs (Thomson et al., “EmbryonicStem Cell Lines Derived From Human Blastocysts,” Science 282:1145-1147(1998), which is hereby incorporated by reference in its entirety);keratinocyte-derived K04 hiPSCs (Maherali et al., “A High-EfficiencySystem for the Generation and Study of Human Induced Pluripotent StemCells,” Cell Stem Cell 3:340-345 (2008), which is hereby incorporated byreference in its entirety); and fibroblast-derived C14 and C27 hiPSCs(Chambers et al., “Highly Efficient Neural Conversion of Human ES andiPS Cells by Dual Inhibition of SMAD Signaling,” Nat. Biotechnol.27:275-280 (2009), which is hereby incorporated by reference in itsentirety). Specific features of several published protocols wereselected for the production of glial progenitor cells from hESCs (Hu etal., “Differentiation of Human Oligodendrocytes From Pluripotent StemCells,” Nat. Protoc. 4:1614-1622 (2009); Izrael et al., “HumanOligodendrocytes Derived From Embryonic Stem Cells: Effect of Noggin onPhenotypic Differentiation in Vitro and on Myelination in Vivo,” Mol.Cell. Neurosci. 34:310-323 (2007), which are hereby incorporated byreference in their entirety) and then optimized to produce the resultanthybrid protocol for use with WA09/H9 hESCs. The resultant protocol wasmodified to further optimize its efficiency with the three hiPSC lines,which were derived in different labs, from different cell sources, andusing different reprogramming protocols (Chambers et al., “HighlyEfficient Neural Conversion of Human ES and iPS Cells by Dual Inhibitionof SMAD Signaling,” Nat. Biotechnol. 27:275-280 (2009); Maherali et al.,“A High-Efficiency System for the Generation and Study of Human InducedPluripotent Stem Cells,” Cell Stem Cell 3:340-345 (2008), which arehereby incorporated by reference in their entirety) (FIG. 2). Theresultant six-stage OPC differentiation protocol, which spans a range of110-150 days in vitro as described above and schematized in FIG. 1,efficiently generated human OPCs (hOPCs) as well as their matureprogeny, including both astrocytes and OLs, from hESCs and hiPSCs alike(FIGS. 1B-1P). Its efficiency of OPC production, as defined by theincidence of OLIG2+/NKX2.2+ gliogenic (Qi et al., “Control ofOligodendrocyte Differentiation by the Nkx2.2 Homeodomain TranscriptionFactor,” Development 128:2723-2733 (2001); Zhou et al., “The bHLHTranscription Factor O1ig2 Promotes Oligodendrocyte Differentiation inCollaboration with Nkx2.2,” Neuron 31:791-807 (2001); Zhou et al.,“Identification of a Novel Family of Oligodendrocyte Lineage-SpecificBasic Helix-Loop-Helix Transcription Factors,” Neuron 25:331-343 (2000),which are hereby incorporated by reference in their entirety) cellclusters in stage 6, ranged from 45.4±20.3% in WA09/H9-derived hESCs to73.8±8.7%, 78.9±6.1%, and 79.5±8.5% in K04-, C14-, and C27-derived OPCs,respectively (all data are provided as means±SEM; FIGS. 3A and 4). Thus,each of the hiPSC and hESC lines could be directed into highly enrichedpreparations of OLIG2+/PDGFRα+/NKX2.2+/SOX10+ OPCs. Indeed, theefficiencies of OPCs' differentiation from hiPSCs, whether induced fromkeratinocytes (K04 cells) or fibroblasts (C14 and C27 cells), wereconsistently higher than that of WA09/H9 hESCs.

Example 2—Both Astrocytes and OLs are Efficiently Derived fromhiPSC-Derived hOPCs

Both in vitro and in vivo, hiPSC OPCs readily differentiated intoastrocytes as well as OLs. GFAP-defined astroglia first appeared by 70days in vitro (DIV), significantly earlier than OLs did. By late stage6, at 120 DIV, GFAP+ astrocytes were found to be abundant when gliogenicspheres were plated onto a polyornithine/laminin-coated surface (FIGS.3B-3D). By that time, GFAP+ cells comprised 40%-50% of cells inOPC-induced cultures, across all cell lines (FIG. 3D). QuantitativeRT-PCR confirmed the upregulation of GFAP messenger RNA (mRNA)expression during OPC differentiation in all cell lines (Table 2).

TABLE 2 Astrocytic appearance during OPC induction: qPCR of GFAP hiPSClines Stage I n Stage 6 n C27 1.0 ± 0.1% 4 15,801.8 ± 7393.7%  4 K04 1.0± 0.1% 3 9,623.6 ± 2434.6% 5 WA09/H9 1.0 ± 0.1% 5 5,077.1 ± 3526.9% 3 %± SEM Human iPSC cultures were subjected to quantitative real-time PCR(qPCR) for astrocytic glial fibrillary acidic protein (GFAP) (normalizedto GAPDH), as a dual function of cell line and stage of OPCdifferentiation in vitro. All data are provided as means ± SEM.

The production of OLs from hESC and hiPSC-derived OPCs was triggered bythe withdrawal of gliogenic growth factors to half-normal levels (seeMaterials and Methods). When hiPSC OPCs were exposed to those conditionsfor 2 weeks, a proportion matured into 04+ and/or myelin basic protein(MBP)+ OLs (FIGS. 3E-3G). According to flow cytometry, 04+ OLs in C27,C14, and K04 hiPSC-derived OPC cultures respectively comprised11.9±3.8%, 4.1±0.9%, and 7.6±1.5% of all cells (at 194±15, 186±14, and205±14 DIV, respectively; means±SEM) (FIG. 5A; Table 3). Of note, theculture conditions favored initial oligodendrocytic differentiation, butnot postmitotic oligodendrocytic survival, because the focus was onpreparing populations of transplantable lineage-biased progenitors andimmature oligodendroglia rather than more mature—but lesstransplantable—process-bearing OLs.

TABLE 3 Flow cytometric delineation of hiPSC oligodendroglial abundancein vitro hiPSC lines O4+ Average DIV C27 11.9 ± 3.8%  194.3 ± 15   C144.1 ± 0.9% 186.0 ± 13.6 K04 7.6 ± 1.5% 204.9 ± 14.0 n = 4-7  % ± SEMHuman iPSC-derived OPCs and early oligodendroglia from different celllines (C27, C14 and K04) were collected and stained for oligodendrocyticsulfatide, as recognized by MAb O4, late in at stage 6 (>120 days invitro, DIV), then analyzed by flow cytometry. Results are given asproportions (mean percentages ± SEM) of O4+ cells; N = 4-7 repeats/cellline.

Example 3—OPCs could be Isolated from hiPSC Cultures by CD140a- andCD9-Directed Fluorescence-Activated Cell Sorting

Flow cytometry for A2B5, CD140a/PDGFaR, and the tetraspanin CD9 (Berryet al., “Cytology and Lineage of NG2-Positive Glia,” J. Neurocytol.31:457-467 (2002); Terada et al., “The Tetraspanin Protein, CD9, IsExpressed by Progenitor Cells Committed to Oligodendrogenesis and IsLinked to Betal Integrin, CD81, and Tspan-2,” Glia 40:350-359 (2002),which are hereby incorporated by reference in their entirety) was nextused for identifying and quantifying hiPSC OPCs in stage 6 culture(FIGS. 5B and 5C). CD140a+ OPCs derived from C27, C14, and K04 hiPSCsrespectively comprised 33.0±10.3%, 32.8±12.0%, and 41.1±6.1% of allcells, compared to 37.5±10.2% of H9-derived cells (FIG. 5C; Table 4).The CD9+ fraction of CD140a+ cells, which defined a later-stage pool ofOPCs, comprised 24.0±8.0% and 12.4±2.3% of cells in stage 6 C27 and K04hiPSC cultures, respectively; matched cultures of H9-derived OPCsincluded 15.0±4.9% CD9+/CD140a+ cells (FIG. 5C; Table 4; n=4-7 repeatseach). Thus, hiPSC OPCs could be identified and isolated at differentstages of lineage restriction, which were serially represented by A2B5,CD140a, CD9, and 04. Selection based on these epitopes permits theisolation of relatively pure populations of hiPSC OPCs while removingresidual undifferentiated cells from the isolate.

TABLE 4 Flow cytometry of cell-selective surface markers during OPCinduction hiPSC lines CD140a⁺ CD9⁺ CD140a⁺/CD9⁺ A2B5⁺ Average DIV C2733.0 ± 10.3% 40.5 ± 5.6% 24.0 ± 8.0% 67.1 ± 12.5% 168.7 ± 14   C14 32.8± 12.0% 28.5 ± 5.3% 16.2 ± 7.0% 31.5 ± 20.3% 165.8 ± 11   K04 41.1 ±6.1%  19.3 ± 3.6% 12.4 ± 2.3% 25.6 ± 4.9%  177.2 ± 12.7 W09/H9 37.5 ±10.2% 22.3 ± 5.3% 15.0 ± 4.9% 15.0 ± 4.9%  148.2 ± 19.5 n = 4-7  % ± SEMThe OPCs derived from 3 different hiPSC cell lines (C27, C14 and K04),as well as from hESCs (WA9/H9), were collected and stained for CD140a,CD9, or A2B5 late in stage 6 (>120 DIV), then analyzed by flowcytometry. Data include the average proportion (mean ± SEM) of CD140a₊,CD9₊, CD140a₊/CD9₊ and A2B5₊ cells (n = 4 to 7 repeats/cell; mean ±SEM).

Example 4—hiPSC-Derived OLs Generate MBP in Contact with Human Axons InVitro

The ability of hiPSC OLs to myelinate axons in vitro was examined. hiPSCOPCs from each cell line were cocultured gestational age (g.a.) fetalbrain using polysialyted neural cell adhesion molecule(PSA-NCAM)-directed selection (Windrem et al., “Neonatal ChimerizationWith Human Glial Progenitor Cells Can Both Remyelinate and Rescue theOtherwise Lethally Hypomyelinated Shiverer Mouse,” Cell Stem Cell2:553-565 (2008), which is hereby incorporated by reference in itsentirety). The neurons were cultured on laminin for 10-14 days to allowphenotypic maturation and fiber extension and were confirmed to be freeof tissue-derived OLs by 04 immunolabeling. hiPSC OPCs were thenprepared as clusters of 50-100 mm in diameter and cocultured with thefetal neurons for 4 weeks; the cultures were then immunolabeled for MBPand neurofilament (NF). Confocal imaging revealed abundant MBP+processes that contacted axons and initiated ensheathment (FIGS. 3H-3J),though unambiguous myelin formation was not noted at the time pointsimaged. Thus, to better assess myelinogenesis by hiPSC OPCs, theirengraftment and myelination in vivo was evaluated.

Example 5—hiPSC OPCs Efficiently and Functionally Myelinate the ShivererBrain

To definitively establish the myelination competence of hiPSC OPCs,these cells were transplanted into newborn homozygous shiverer(shi/shi)x rag2^(−/−) immunodeficient mice. For this experiment, themice were implanted with 100,000 hiPSC-derived OPCs bilaterally into thecorpus callosum (n=4-7 mice per hiPSC line for K04, C27, and C14hiPSC-derived OPCs), using previously described methods (Windrem et al.,“Neonatal Chimerization With Human Glial Progenitor Cells Can BothRemyelinate and Rescue the Otherwise Lethally Hypomyelinated ShivererMouse,” Cell Stem Cell 2:553-565 (2008), which is hereby incorporated byreference in its entirety). At 3 or 4.5 months of age, the mice werekilled and their brains were analyzed in terms of donor celldistribution and density, myelin production and the proportion ofmyelinated axons, and nodal reconstitution. All three of the hiPSCline-derived OPCs were able to robustly myelinate the recipient brains;from each line, high donor cell densities and widespread dispersal wereobserved throughout the forebrain white matter (FIGS. 6A and 6B). C27,C14, and K04 hiPSC OPC-derived oligodendrocytic differentiation andmyelination were analogous in extent, with robust myelination of thecorpus callosum and capsules (FIGS. 6C, 6E, 6H, 7B, 7G, and 7J). As aresult of the superior initial neutralization of these two linesrelative to C14, higher net yields of OPCs were achieved with C27 andK04 hiPSCs and hence quantitative assessment of myelination inrecipients of C27 or K04 hiPSC OPCs was pursued.

Quantitative histology revealed that within the corpus callosa of 3month (13 week)-old shiverer recipients, C27 and K04 hiPSC-derived OPCsand oligodendroglia, defined as human nuclear antigen (hNA)⁺/OLIG2⁺,achieved densities of 29,498±13,144 and 37,032±8,392 cells/mm³,respectively. Among these, 7,298±2,659 (C27) and 2,328±650 (K04)cells/mm³ expressed MBP; these comprised 10.9±5.1% (C27) and 4.7±1.1%(K04) of all donor cells within the sampled midline of the corpuscallosum at the 13 week time point. To assess the myelination efficiencyin terms of the proportion of axons myelinated, confocal analysis wasused to quantify the fraction of callosal axons ensheathed by hiPSColigodendroglia in the three mice engrafted with C27 hiPSC-derived OPCs.At the 13 week time point analyzed, 17.2±7.2% of host mouse axons wereensheathed within the three sampled callosa (FIG. 7B). Remarkably, thedensity of hiPSC-OPC donor derived myelination and the proportion ofensheathed axons at 13 weeks proved as high as, and exceeded, thoseachieved by OPCs derived from second-trimester fetal brain tissue,whether isolated as A2B5⁺/PSA-NCAM⁻ or CD140a⁺ cells (Sim et al.,“CD140a Identifies a Population of Highly Myelinogenic,Migration-Competent and Efficiently Engrafting Human OligodendrocyteProgenitor Cells,” Nat. Biotechnol. 29:934-941 (2011); Windrem et al.,“Fetal and Adult Human Oligodendrocyte Progenitor Cell IsolatesMyelinate the Congenitally Dysmyelinated Brain,” Nat. Med. 10:93-97(2004); Windrem et al., “Neonatal Chimerization With Human GlialProgenitor Cells Can Both Remyelinate and Rescue the Otherwise LethallyHypomyelinated Shiverer Mouse,” Cell Stem Cell 2:553-565 (2008), whichare hereby incorporated by reference in their entirety).

Example 6—hiPSC OPCs Efficiently Generate Astrocytes and OLs In Vivo

Besides the large numbers of hiPSC OPCs that differentiated asmyelinogenic OLs in the shiverer mouse brain, large numbers alsoremained as resident OLIG2⁺ and NG2⁺ progenitor cells, and many OPCs ofall three hiPSC lines differentiated as astrocytes as well, particularlyas fibrous astrocytes of the white matter (FIGS. 6D, 6F, and 6I). WhenhiPSC-OPC-transplanted mice were assessed at 13 weeks after neonatalgraft, most donor cells persisted as progenitors or had initiatedoligodendroglial differentiation; by that time point, the net proportionof OLIG2⁺ cells, which included both OPCs and oligodendroglia, arisingfrom all K04 and C27 transplanted cells was 78.7±2.4%, whereas theremainder were largely donor-derived GFAP+ astroglia (Table 5 below).

Interestingly, despite the widespread infiltration of the recipientbrains by hiPSC OPCs, substantial astrocytic differentiation was notedby those cells within the presumptive white matter, within which thedonor cells differentiated as morphologically apparent fibrousastrocytes, in close association with hiPSC derived OLs. ThesehiPSC-derived astrocytes might have been generated fromlineage-restricted hiPSC-derived astrogliogenic precursors or byastrocytic differentiation in situ from still-bipotential hOPCs. Ineither case, by 3 months after neonatal transplant, the callosal andcapsular white matter of shiverer recipients of OPC grafts derived fromall three hiPSC lines manifested human astrocytic scaffolds harboringdensely engrafted myelinogenic OLs, in each case yielding substantiallyreconstructed and densely myelinated central white matter (FIGS. 6D, 6F,and 6I).

TABLE 5 Phenotypic Differentiation of hiPSC-OPC Derivatives In Vivo at13 Weeks of Age C27—13 weeks (n = 4) K04—13 week (n = 5) Marker Mean ±SEM Mean ± SEM OLIG2 68.0 ± 9.5% 87.2 ± 9.9% MBP 12.0 ± 3.8%  4.7 ± 1.1%hGFAP 11.6 ± 5.1%  0.9 ± 0.5% Ki67  8.5 ± 2.9% 12.6 ± 3.2%Immunolabeling of engrafted mice at 13 weeks of age revealed that amajority of engrafted hiPSC OPCs and their progeny remained asOLIG2₊/hGFAP⁻ MBP⁻ OPCs; nonetheless, significant complements ofhiPSC-derived MBP₊ oligodendroglia were noted in the engrafted mice, aswere hGFAP+astrocytes, especially in OPCs derived from the C27 line. TheKi67 index at 13 weeks was relatively high, but no higher than that ofneonatally-delivered fetal tissue-derived OPCs at the same postnatalage.

Example 7—Neonatal Engraftment with hiPSC OPCs could Rescue the ShivererMouse

Whether the robust engraftment and myelination noted in transplantedshiverer mice were sufficient to ameliorate neurological deteriorationand prolong the survival of shiverer mice, which typically die by 20weeks of age, was assessed. To this end, a set of 22 neonatal homozygousshiverer x rag2 nulls were transplanted with 300,000 C27-derived hiPSCOPCs using a five-site forebrain and brainstem injection protocol thatachieves whole-neuraxis engraftment via transplanted OPCs (FIGS. 8A-8D)(Windrem et al., “Neonatal Chimerization With Human Glial ProgenitorCells Can Both Remyelinate and Rescue the Otherwise LethallyHypomyelinated Shiverer Mouse,” Cell Stem Cell 2:553-565 (2008), whichis hereby incorporated by reference in its entirety). A matched set of19 littermate controls were injected only with saline, and both setswere housed without further manipulation. Predictably, the 19unimplanted shiverer controls died before 5 months of age, with a mediansurvival of 141 days. In contrast, 19 of the 22 implanted mice livedlonger than the longest-lived control mouse. The transplanted miceexhibited greatly prolonged survival (FIG. 8E), with reduced death overthe 9 month period of observation, after which the experiment wasterminated so that surviving mice could be processed for bothimmunohistochemical assessment of late-stage myelination and nodalreconstitution and for ultrastructural analysis (see Example 8).Comparison of the Kaplan-Meier survival plots of transplanted andcontrol mice revealed a highly significant difference (chi square=17.95by the Gehan-Breslow-Wilcoxon test; p<0.0001) (FIG. 8E). Thosetransplanted mice that survived beyond 6 months uniformly exhibitedsubstantial myelination of the brain, brainstem, and cerebellum (FIGS.8A-8D and 9). Remarkably, the time-point-matched degree of cerebralmyelination, as well as the proportion of shiverers alive at any giventime point, was greater in hiPSC-OPC-engrafted mice than in micepreviously engrafted with fetal-human-tissuederived, A2B5-sorted OPCs(Windrem et al., “Neonatal Chimerization With Human Glial ProgenitorCells Can Both Remyelinate and Rescue the Otherwise LethallyHypomyelinated Shiverer Mouse,” Cell Stem Cell 2:553-565 (2008), whichis hereby incorporated by reference in its entirety), which hadotherwise been treated identically.

Example 8—hiPSC OPCs Generated Ultrastructurally Mature Myelin withNodal Reconstitution

In light of the markedly extended survival of hiPSC-OPC-transplantedmice, whether this was associated with the formation ofultrastructurally compact myelin around host axons by hiPSC OLs wasexamined. To this end, electron microscopy was used on samples of corpuscallosum derived from 22- to 36-week-old engrafted shiverers (n=3).These mice comprised animals that had been subjected to the five-siteinjection protocol and survived significantly longer than theirunengrafted controls; these apparently rescued mice were killed afterrelatively long survival time points to permit assessment of theirmyelin integrity and quality. The recipient callosa were denselymyelinated by mature compact myelin characterized by concentricallyorganized major dense lines (FIGS. 10A-10E) and interlaminar tightjunctions (FIGS. 10F, 10G, and 11C); and the engrafted callosa werequite unlike those of their untransplanted shiverer controls, whichfailed to exhibit major dense lines or any other evidence of myelincompaction (FIGS. 11B, 11D, and 11E).

Anatomic reconstitution of nodes of Ranvier was also noted in thesemice, as determined by immunolabeling of Caspr and βIV spectrin, whichrespectively identified paranodal and nodal segments of newly myelinatedaxons (FIGS. 10H and 10I). In past studies, the anatomic and antigenicreconstitution of nodal architecture was correlated with the restorationof both rapid conduction and functional competence (Windrem et al.,“Neonatal Chimerization With Human Glial Progenitor Cells Can BothRemyelinate and Rescue the Otherwise Lethally Hypomyelinated ShivererMouse,” Cell Stem Cell 2:553-565 (2008), which is hereby incorporated byreference in its entirety). The rapid and robust reacquisition of nodalarchitecture in these mice indicates that hiPSC-derived OLs generate thecues necessary for the nodal organization of axonal proteins, upon whichthe formation of functional nodes of Ranvier depends.

Together, these data indicate that hiPSC-derived OPCs can efficientlygenerate OLs, which in turn can robustly myelinate the hypomyelinatedshiverer forebrain, and that the myelin thereby generated is able torestore nodal architecture as well as to ensheath axons as efficientlyas purified isolates of fetal-tissue-derived OPCs.

Example 9—hiPSC OPCs were Nontumorigenic In Vivo

The persistence of undifferentiated pluripotent stem cells may causeeither teratomas or neuroepithelial tumors in graft recipients (Roy etal., “Functional Engraftment of Human ES Cell-DerivedDopaminergicneurons Enriched by Coculture With Telomerase-ImmortalizedMidbrain Astrocytes,” Nat. Med. 12:1259-1268 (2006), which is herebyincorporated by reference in its entirety). To assess whether anypluripotent or incompletely differentiated hESCs or hiPSCs remained innominally fully differentiated OL cultures, both immunolabeling andqRT-PCR were used to assess the expression of pluripotent markers bylate-stage hiPSC-derived OPCs. By 100 DIV, no detectable OCT4, NANOG, orSSEA4 protein could be found in OPCs derived from any of the hESC andhiPSC lines used in this study. Similarly, qRT-PCR revealed thattranscripts of OCT4 and human telomerase reverse transcriptase (hTERT)were downregulated to essentially undetectable levels by 95 or more DIV(FIGS. 4J and 4K). The in vivo expression of OCT4, NANOG, and SSEA4 byengrafted OPCs 3 months after transplantation was examined. As noted,only a small minority of hNA+ donor cells were unstained by OLIG2, MBP,or GFAP. Many donor-derived cells expressed nestin or SOX2, indicatingtheir persistence as neural progenitors, but no persistent expression ofOCT4, NANOG, or SSEA4 was detectable in any of these cells, from any ofthe lines assessed.

Accordingly, no evidence of teratoma formation was found in any of the16 shi/shi x rag2^(−/−) mice examined for this purpose, which includedmice transplanted neonatally with 100,000 cells and killed either 3(n=11) or 4.5 (n=5) months later. Available mice in the survival serieswere also examined, all of whom had been transplanted at five sites witha total of 300,000 cells and had died between 4 and 9 months of age(n=10); none had any evidence of teratomas, heterotopias, or any type oftumor formation. In addition, hiPSCs were transplanted into normallymyelinated rag2-null mice to assess tumorigenicity in the wild-typemyelin environment as well. Of five mice examined 6 months aftertransplantation, none showed any evidence of tumor formation,heterotopias, or even foci of undifferentiated expansion. Of note,persistent expression of SOX2, KLF4, and c-MYC mRNA was noted by qPCR inthe hiPSC-derived cells, reflecting some level of unsilenced expressionof the lentivirally inserted reprogramming genes; nonetheless, theexpression of the these transcripts was not associated withtumorigenesis by cells transplanted at the end of stage 6.

The lack of tumor formation in hiPSC-OPC-engrafted mice was associatedwith a significant decrease in the mitotic fraction of the implantedhiPSC OPCs as a function of time after graft. hiPSC OPC proliferation invivo was measured as Ki67 expression by all human donor cells, which wasnoted to decrease linearly from 3 months (13.6±0.6%) to 6 months(4.3±0.04%) of age (R2=0.9; p=0.001; n=7).

To establish the role of the differentiation protocol in diminishing therisk of tumorigenesis, rag2-null mice were also transplanted with bothC27 and K04 hiPSCs at the end of stages 1 and 3. This was also done as apositive control for tumor detection, given the lack of observed tumorsin the hiPSC OPC (stage 6)-engrafted mice, as much as 9 months aftertransplant. Yet in contrast to the hiPSC-OPC-engrafted mice, which wereentirely tumor-free, every animal engrafted with earlier-stage hiPSCsmanifested histologically overt tumor formation by 3 months (n=8 miceengrafted with stage 1 hiPSCs; n=6 with stage 3 cells). Thus, thedifferentiation protocol appeared to effectively deplete the donor cellpool of persistent undifferentiated cells; the resultant grafts of hiPSCOPCs proved uniformly nontumorigenic when studied as long as 9 monthsafter transplant.

Discussion of Examples 1-9

In this study, the feasibility of using hiPSCs to generate highlyenriched populations of both astrocytes and myelinogenic central OLs,with high efficiency and yield has been established. The success of theprotocol described herein in all four lines used in this study, whichinclude WA09/H9 hESCs and K04, C14, and C27 iPSCs, indicates its broadapplicability, and the highly efficient gliogenesis afforded by thisstrategy indicates its robust nature. Most importantly, the robustmyelination that was noted in vivo, which compared favorably to thatpreviously demonstrated by tissue derived fetal human glial progenitors,indicated the probable functional integration and utility of thesegrafts. Accordingly, it was noted that myelination-deficient shiverersengrafted neonatally with hiPSC OPCs survived substantially longer thandid both their untransplanted and saline-injected controls; indeed, overthree-fourths of hiPSC-OPC-transplanted mice survived over 6 months,long after all untreated control mice had died. As a result, hiPSC OPCsfrom single-patient skin samples can now be reliably produced insufficient numbers to provide myelinogenic autografts largely, thoughperhaps not completely (Zhao et al., “Immunogenicity of InducedPluripotent Stem Cells,” Nature 474:212-215 (2011), which is herebyincorporated by reference in its entirety), free of rejection risk.

Importantly, the myelination efficiency of the implanted iPSC derivedOPCs, defined as the proportion of central axons myelinated as afunction of time after graft, proved as high as that which was hadpreviously achieved using tissue-derived, CD140a sorted OPCs (Sim etal., “CD140a Identifies a Population of Highly Myelinogenic,Migration-Competent and Efficiently Engrafting Human OligodendrocyteProgenitor Cells,” Nat. Biotechnol. 29:934-941 (2011), which is herebyincorporated by reference in its entirety). Indeed, it was remarkable tonote that the proportion of axons ensheathed was as high in enriched butunsorted hiPSC-OPC grafts as in fetal-tissue-derived OPC grafts that hadbeen sorted for CD140a+ cells prior to transplant. Indeed, thehiPSC-OPCs grafts myelinated more axons more rapidly than didA2B5+/PSA-NCAM-sorted fetal-tissue derived cells, probably reflectingthe higher proportion of bipotential glial progenitor cells in thehiPSC-OPC populations by the time of their harvest and transplantation.

In light of the robust myelination afforded by hiPSC-OPC grafts, it wasasked whether neonatal transplantation of hiPSC OPCs might be sufficientto rescue the phenotype and survival of recipient shiverer homozygotes,as had previously been observed in a minority of shiverers transplantedwith fetal-human brain-derived OPCs. The hiPSC-OPC-transplanted miceindeed exhibited markedly improved survival; death was both delayed andreduced overall in the transplanted group over the 9 month period ofobservation. As previously documented with fetal-brain-tissue-derivedOPC grafts, the rescued mice manifested progressive resolution of theirneurological deficits (Windrem et al., “Neonatal Chimerization WithHuman Glial Progenitor Cells Can Both Remyelinate and Rescue theOtherwise Lethally Hypomyelinated Shiverer Mouse,” Cell Stem Cell2:553-565 (2008), which is hereby incorporated by reference in itsentirety). Remarkably, however, the proportion of animals whose survivalbenefitted from hiPSC-OPC transplantation was substantially higher thanthat which was previously reported using tissue-derived human OPCs:whereas it was observed that only one-quarter of shiverer micetransplanted with tissue-derived OPCs survived beyond 6 months of age(Windrem et al., “Neonatal Chimerization With Human Glial ProgenitorCells Can Both Remyelinate and Rescue the Otherwise LethallyHypomyelinated Shiverer Mouse,” Cell Stem Cell 2:553-565 (2008), whichis hereby incorporated by reference in its entirety), in the presentstudy over half of the hiPSC-OPC-engrafted mice did so (FIG. 8E).Nonetheless, some later deaths beyond 7 months of age were still noted;this may reflect an inhomogeneous dispersal of hiPSC OPCs that wasobserved in some animals, the nature of which is under investigation.Those late deaths notwithstanding, at least one-fifth of the miceappeared to represent outright clinical rescues, though these survivorswere sacrificed at ≧9 months for histological and ultrastructuralanalysis. These provocative data demonstrate the superiority of hiPSCOPCs as therapeutic vectors, perhaps by virtue of their more rapidmyelinogenesis, which may be a function of the prolonged differentiationconditions that was employed in this OPC induction protocol.

Interestingly, no evidence of tumorigenesis from implanted hiPSC-derivedglial progenitors was observed at time points as long as 9 months aftertransplant. This was surprising, given that previous studies hadprovided ample evidence for the risk of tumor formation from eitherresidual undifferentiated cells (Pruszak et al., “CD15, CD24, and CD29Define a Surface Biomarker Code for Neural Lineage Differentiation ofStem Cells,” Stem Cells 27:2928-2940 (2009), which is herebyincorporated by reference in its entirety) or from partiallydifferentiated neuroepithelial cells in hESC-derived transplants (Roy etal., “Functional Engraftment of Human ES Cell-DerivedDopaminergicneurons Enriched by Coculture With Telomerase-ImmortalizedMidbrain Astrocytes,” Nat. Med. 12:1259-1268 (2006), which is herebyincorporated by reference in its entirety). It is possible that theprolonged differentiation protocols employed to produce OPCs are robustenough to effectively eliminate any residual undifferentiated cellsprior to transplantation. It is similarly possible that epigenetic markspersisting in reprogrammed hiPSCs effectively lowered the later risk oftumorigenesis by their differentiated derivatives. In any case, evenlonger survival time points will be needed, with more animals and anintensive search for any residual undifferentiated and/or potentiallytumorigenic cells in vivo, before one can confidently state the safetyof these grafts. Should tumorigenesis at any point be a concern, thenhiPSC OPCs may be sorted to purity before transplantation, on the basisof the high incidence of definitively pro-oligodendrocytic CD9⁺/CD140a⁺cells in the cultures, and the ability to isolate these cells byfluorescence-activated cell sorting (FACS) based upon these coexpressedepitopes (Sim et al., “CD140a Identifies a Population of HighlyMyelinogenic, Migration-Competent and Efficiently Engrafting HumanOligodendrocyte Progenitor Cells,” Nat. Biotechnol. 29:934-941 (2011),which is hereby incorporated by reference in its entirety).

These findings indicate that high-efficiency in vivo oligodendrocyticdifferentiation and myelination can be achieved from hiPSCs, indicatingthe utility of iPSC-derived autografts in treating acquired disorders ofmyelin. Yet it is also important to note the efficient,context-dependent generation of both fibrous and protoplasmic astrocytesfrom engrafted hiPSC OPCs. Besides the importance of astroglia ineffecting the structural and physiological reconstitution ofdysmyelinated tracts, astrocytic engraftment may be of particularimportance in correcting dysmyelinating disorders of enzyme deficiency,given that astrocytic lysosomal enzymes have been found to readilytransit from wild-type to deficient glia within brain tissue, in amanner potentially sufficient to rescue enzyme-deficient hosts (Lee etal., “Stem Cells Act Through Multiple Mechanisms to Benefit Mice WithNeurodegenerative Metabolic Disease,” Nat. Med. 13:439-447 (2007), whichis hereby incorporated by reference in its entirety). In addition,hiPSC-derived astrocytes may prove to be critically importanttherapeutic vectors for diseases of primarily astrocytic pathology(Krencik et al., “Specification of Transplantable Astroglial SubtypesFrom Human Pluripotent Stem Cells,” Nat. Biotechnol. 29:528-534 (2011),which is hereby incorporated by reference in its entirety), such asAlexander disease and the vanishing white-matter disorders (Bugiani etal., “Defective Glial Maturation in Vanishing White Matter Disease,” J.Neuropathol. Exp. Neurol. 70:69-82 (2011), which is hereby incorporatedby reference in its entirety), in which myelin loss occurs but may besecondary to astrocytic dysfunction. In each of these cases, however,the therapeutic use of iPSC-derived astroglia will need to be pairedwith methods for the ex vivo correction of the genetic defectscharacteristic of these disorders.

Human iPSC OPCs might thus be attractive vectors for restoring orreplacing glial populations in a variety of disease settings. Mostcritically, the data presented herein indicates the preferential use ofhiPSC-derived OPCs to restore lost myelin in disorders such as multiplesclerosis and traumatic demyelination, in which no genetic abnormalitiesmight complicate the use of a patient's own somatic cells as the iPSCsource. iPSC OPCs may similarly prove of great therapeutic value ingenetic disorders of myelin, such as Pelizaeus-Merzbacher disease,recognizing that the underlying genetic defect must first be repaired inthe donor somatic cells before glial progenitor induction andimplantation. The present study thus establishes the technicalfeasibility and efficacy of generating myelinogenic OLs from hiPSCs andindicates the clinical situations in which this approach might be mostappropriate. The clinical application of patient-specific, somaticcell-derived glial progenitor cell transplants for the treatment ofacquired disorders of myelin, as well as of the broader spectrum ofhuman glial pathologies can now be reasonably contemplated.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

What is claimed is:
 1. A method of producing an enriched preparation ofoligodendrocyte progenitor cells said method comprising: culturing apopulation of induced pluripotent stem cells under conditions effectivefor said cells to form embryoid bodies; inducing cells of the embryoidbodies to differentiate into neuroepithelial cells and formneuroepithelial cell colonies; and exposing the neuroepithelial cellcolonies to conditions effective to induce differentiation tooligodendrocyte progenitor cells, thereby forming an enrichedpreparation of oligodendrocyte progenitor cells co-expressing OLIG2 andCD140α/PDGFRα.
 2. The method of claim 1, wherein the oligodendrocyteprogenitor cells of the enriched preparation further express SOX10, CD9,or a combination thereof.
 3. The method of claim 1 further comprising:separating, from the enriched preparation of oligodendrocyte progenitorcells, CD140α/PDGFRα positive cells thereby producing a purifiedpreparation of CD140α/PDGFRα expressing oligodendrocyte progenitorcells.
 4. The method of claim 1, wherein said neuroepithelial cellsco-express PAX6 and SOX1.
 5. The method of claim 1, wherein the inducedpluripotent stem cells are human induced pluripotent stem cells.
 6. Themethod of claim 1 further comprising: reprogramming a population ofnon-pluripotent cells to a pluripotent state thereby forming thepopulation of induced pluripotent stem cells.
 7. The method of claim 6,wherein the population of non-pluripotent cells comprise a population ofskin cells, umbilical cord blood cells, peripheral blood cells, or bonemarrow cells.
 8. The method of claim 1, wherein said exposing comprises:converting the neuroepithelial cells to pre-oligodendrocyte progenitorcells which are OLIG2+/NKX2.2−; transforming the OLIG2+/NKX2.2−pre-oligodendrocyte progenitor cells to OLIG2+/NKX2.2+pre-oligodendrocyte progenitor cells; and forming the enrichedpopulation of oligodendrocyte progenitor cells from the OLIG2+/NKX2.2+pre-oligodendrocyte progenitor cells.
 9. The method of claim 1 furthercomprising: differentiating the enriched preparation of oligodendrocyteprogenitor cells into oligodendrocytes and/or astrocytes.
 10. Thepreparation produced by the method of claim
 1. 11. The preparationproduced by the method of claim
 3. 12. The preparation produced by themethod of claim
 9. 13. A method of treating a subject having a conditionmediated by a loss of myelin or a loss of oligodendrocytes, said methodcomprising: administering to the subject the preparation of claim 10under conditions effective to treat the condition.
 14. The method ofclaim 13, wherein the preparation is administered to one or more sitesof the brain, the brain stem, the spinal cord, or a combination thereof.15. The method of claim 14, wherein the preparation is administeredintraventricularly, intracallosally, or intraparenchymally.
 16. Themethod of claim 13, wherein the preparation is derived from saidsubject.
 17. The method of claim 13, wherein said administering iscarried out under conditions effective to achieve whole-neuraxisengraftment by the oligodendrocyte progenitor cells of the preparation.18. The method of claim 13, wherein the condition is an autoimmunedemyelination condition.
 19. The method of claim 18, wherein theautoimmune demyelination condition is selected from the group consistingof multiple sclerosis, neuromyelitis optica, transverse myelitis, andoptic neuritis.
 20. The method of claim 13, wherein the condition is avascular leukoencephalopathy.
 21. The method of claim 20, wherein thevascular leukoencephalopathy is selected from the group consisting ofsubcortical stroke, diabetic leukoencephalopathy, hypertensiveleukoencephalopathy, age-related white matter disease, and spinal cordinjury.
 22. The method of claim 13, wherein the condition is a radiationinduced demyelination condition.
 23. The method of claim 13, wherein thecondition is a pediatric leukodystrophy.
 24. The method of claim 23,wherein the pediatric leukodystrophy is selected from the groupconsisting of Pelizaeus-Merzbacher Disease, Tay-Sach Disease, Sandhoff'sgangliosidoses, Krabbe's disease, metachromatic leukodystrophy,mucopolysaccharidoses, Niemann-Pick A disease, adrenoleukodystrophy,Canavan's disease, Vanishing White Matter Disease, and AlexanderDisease.
 25. The method of claim 13, wherein the condition isperiventricular leukomalacia or cerebral palsy.